KR101899835B1 - Anti-lrp6 antibodies - Google Patents

Anti-lrp6 antibodies Download PDF

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KR101899835B1
KR101899835B1 KR1020127027566A KR20127027566A KR101899835B1 KR 101899835 B1 KR101899835 B1 KR 101899835B1 KR 1020127027566 A KR1020127027566 A KR 1020127027566A KR 20127027566 A KR20127027566 A KR 20127027566A KR 101899835 B1 KR101899835 B1 KR 101899835B1
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amino acid
hvr
acid sequence
antibody
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KR20130012256A (en
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에릭 부리스
릭 카라노
안드레아 코치란
마이크 코스타
베니타 데 알메이다
제임스 에른스트
얀 공
라미 한누쉬
폴 폴라키스
본니 루빈펠드
마크 솔로웨이
얀 유
팀 크리스토퍼 카오
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제넨테크, 인크.
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Priority to PCT/US2011/029508 priority patent/WO2011119661A1/en
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    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
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Abstract

The present invention provides anti-LRP6 antibodies and methods of use thereof. Certain aspects of the invention provide bispecific anti-LRP6 antibodies that inhibit signaling by multiple Wnt isoforms.
[Representative figure]
11B

Description

Anti-LRP6 antibody {ANTI-LRP6 ANTIBODIES}

<Cross reference to related application>

This application claims priority to U.S. Provisional Application No. 61 / 317,137, filed March 24, 2010, and U.S. Provisional Application No. 61 / 394,836, filed October 20, 2010, the disclosures of which are incorporated herein by reference in their entirety .

[0001]

The present invention relates to anti-LRP6 antibodies and methods of use thereof for the treatment of cancer or skeletal disorders.

Similar to most other morphogen and growth factor signaling pathways, mammalian Wnt signaling involves the use of 19 different ligands, 10 receptors, and a number of co-receptors (including LRP5 / 6, Ror1 / 2 and Ryk) And many times during tissue homeostasis (van Amerongen and Nusse, 2009). In addition, different secreted antagonists that bind to Wnt, such as SFRP1 / 2/3/4/5 and WIF1, or LRP5 / 6 (including DKK1 / 2/4 and SOST), regulate the interaction between the ligand and the receptor. These membranes and extracellular proteins and their multiple isoforms provide differential regulation at the level of expression and combinatorial protein interactions. Most Wnt isoforms were found to bind to the co-receptor LRP5 / 6, and the use of LRP5 / 6 specifies normal or β-catechin-dependent, Wnt signaling. Wnt heterodimates LRP5 / 6 and FZD to mediate phosphorylation and Axin binding of the LRP5 / 6 intracellular domain (Tamai et al., 2000; Semenov et al., 2001; Tamai et al., 2004 ]). DVL is introduced into the complex by direct coupling to both Axin and FZD, and DVL oligomerization can expand these protein complexes on the cytoplasmic surface of the membrane, isolating GSK3 and inhibiting its phosphorylation and &lt; RTI ID = 0.0 &gt;2008; Weng et al., 2008; Zeng et al., 2008; Zeng et al., 2008; , 2009]).

A number of characteristic ligand isoforms characteristic of significant primary sequence diversity that mediate mammalian normal Wnt signaling are in contrast to pairs of highly homologous co-receptors. The LRP6 and LRP5 extracellular domains consist of four homologous domains, designated Nos. E1 through E4, from the N-terminus to the C-terminus, which contain the YWTD-type [beta] -propeller and the EGF-like domain, respectively (Jeon et al., 2001). Each repetition at similar positions in LRP6 and LRP5 is highly conserved, while different repeats in the same protein are considerably more diverse. Interestingly, Bouris et al. (2010) demonstrated that Wnt9b binds exclusively in the E1-E2 region in vitro while Wnt3a binds only to the fragment containing E3-E4, indicating that each repeat or combination of two adjacent repeats is a Wnt iso Lt; / RTI &gt; different subsets of the type. This arrangement will accommodate the diversity of Wnt proteins and will likely allow differential control of these by LRP5 / 6 antagonist ligands. In the Notch and VEGF receptors, their extracellular regions contain repeats of the EGF-like and Ig domains, respectively, and the binding of a plurality of ligand isoforms results in one or two repeats, even though the presence of other repeats can promote binding In the same region of the region. (Rebay et al., 1991; Davis-Smyth et al., 1996; Cunningham et al., 1997).

In the case of receptor tyrosine kinases, ligand-induced dimerization initiates stimulation of kinase activity and signal transduction. Ligand-induced receptor-co-receptor heterodimerization is required for normal Wnt signaling, whereas the role for LRP5 / 6 or FZD homodimerization is not clearly defined. Forced dimerization of different recombinant LRP6 proteins can activate or inhibit Wnt signaling.

beta -catenin-dependent Wnt signaling is initiated by Wnt isoformic binding to both the receptor FZD and the co-receptor LRP5 / 6, which then assembles the multimeric complexes in the cytoplasmic membrane facing surface to mobilize and inactivate kinase GSK3 . Whether or not the mechanically different interactions between Wnt isoforms and receptors regulate this process remains to be determined.

<Overview>

One aspect of the invention provides an isolated antibody that binds to LRP6 that inhibits signaling induced by the first Wnt isoform and enhances signal transduction induced by the second Wnt isoform. In one embodiment, the first Wnt isoform is selected from the group consisting of Wnt3 and Wnt3a. In one embodiment, the second Wnt isoform is selected from the group consisting of Wnt 1, 2, 2b, 4, 6, 7a, 7b, 8a, 9a, 9b, 10a and 10b. In another embodiment, the first Wnt isoform is selected from the group consisting of Wnt 1, 2, 2b, 6, 8a, 9a, 9b and 10b and the second Wnt isoform is selected from the group consisting of Wnt3 and Wnt3a .

One aspect of the invention provides an antibody that binds to the E3-E4 region of LRP6. Another aspect of the invention provides an antibody that binds to the E1-E2 region of LRP6. Another aspect provides antibodies that bind to two different regions of LRP6, such as the E1-E2 region and the E3-E4 region of LRP6. In one aspect, the antibody inhibits Wnt signaling induced by a combination of Wnt1 and Wnt3a. In one aspect, these antibodies inhibit autocrine Wnt signaling.

One aspect of the invention is an isolated antibody that binds to LRP6 and binds to LRP6 that inhibits signaling induced by Wnt isoforms selected from the group consisting of Wnt3 and Wnt3a and binds to Wnt 1, 2, 2b, 6, 8a, 9a , 9b and 10b to an individual having cancer, comprising administering to the individual an effective amount of an isolated antibody that inhibits signaling induced by Wnt isoforms selected from the group consisting of: &lt; RTI ID = 0.0 &gt;

Another aspect of the invention relates to an isolated antibody that binds to LRP6 and inhibits Wnt3 and Wnt3a-induced signal transduction and binds to LRP6 and is induced by Wnt 1, 2, 2b, 6, 8a, 9a, 9b and 10b Comprising administering to a subject having cancer an effective amount of an isolated antibody that inhibits &lt; RTI ID = 0.0 &gt; a &lt; / RTI &gt;

Another aspect of the invention is an isolated antibody that binds to LRP6 and binds to LRP6 and inhibits Wnt3 and Wnt3a-induced signal transduction and binds to Wnt 1, 2, 2b, 4, 6, 7a, 7b, 8a, 9a, 9b, 10a and 10b, comprising administering to an individual having an cancer an effective amount of an isolated antibody that inhibits signal transduction induced by said agent.

One aspect of the invention provides a method of treating an individual having an osteoporosis, osteoarthritis, fracture and bone lesion, comprising administering to the individual an effective amount of an anti-LRP6 antibody described herein.

Another aspect of the present invention is a method of treating a disease or disorder in a subject by administering an effective amount of an anti-LRP6 antibody described herein and a Wnt isoform to a subject, thereby enhancing Wnt signaling induced by the Wnt isotype Induced Wnt signaling.

In addition, certain anti-LRP6 antibodies comprising a bispecific anti-LRP6 antibody are provided. In one embodiment, the isolated antibody that binds to LRP6 comprises a VH comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13 and SEQ ID NO: In one embodiment, the antibody further comprises a VL comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 10 and SEQ ID NO: 12. In one embodiment, the isolated antibody that binds to LRP6 comprises a VH comprising an amino acid sequence having at least 90% homology to the amino acid sequence of SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13 and SEQ ID NO: In one embodiment, the isolated antibody that binds to LRP6 further comprises a VL comprising an amino acid sequence having at least 90% homology to the amino acid sequence selected from the group consisting of SEQ ID NO: 10 and SEQ ID NO: 12.

In one embodiment, the antibody is an isolated bispecific antibody that binds to two different regions of LRP6, including a VH comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: In one embodiment, the bispecific antibody comprises a first VH comprising the amino acid sequence of SEQ ID NO: 15 and a second VH comprising the amino acid sequence selected from the group consisting of SEQ ID NO: 9, SEQ ID NO: 11 and SEQ ID NO: In one embodiment, the bispecific antibody further comprises a VL comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 10 and SEQ ID NO: 12.

In one embodiment, the bispecific antibody that binds to two different regions of LRP6 comprises an amino acid sequence having at least 90% homology to the amino acid sequence selected from the group consisting of SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: Includes VH. In one embodiment, the bispecific antibody that binds to two different regions of LRP6 comprises a first VH comprising an amino acid sequence having at least 90% homology to the amino acid sequence of SEQ ID NO: 15, a first VH comprising SEQ ID NO: 9, And a second VH comprising an amino acid sequence having at least 90% homology to the amino acid sequence selected from the group consisting of SEQ ID NO: In one embodiment, the bispecific antibody further comprises a VL comprising an amino acid sequence having at least 90% homology to the amino acid sequence selected from the group consisting of SEQ ID NO: 10 and SEQ ID NO: 12.

In one embodiment, the isolated bispecific antibody that binds to two different regions of LRP6 comprises (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 17; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 18; And (c) a first VH domain comprising at least one, at least two or all three VH HVR sequences selected from HVR-H3 comprising the amino acid sequence of SEQ ID NO: 19, and (d) the amino acid sequence of SEQ ID NO: HVR-H1; (e) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 23; And (f) a second VH domain comprising at least one, at least two or all three VH HVR sequences selected from HVR-H3 comprising the amino acid sequence of SEQ ID NO: 24. In one embodiment, the isolated bispecific antibody that binds to two different regions of LRP6 comprises (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 17; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 18; And (c) a first VH domain comprising all three VH HVR sequences from HVR-H3 comprising the amino acid sequence of SEQ ID NO: 19, (d) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 22; (e) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 23; And (f) a second VH domain comprising all three VH HVR sequences from HVR-H3 comprising the amino acid sequence of SEQ ID NO: 24.

In one embodiment, the isolated bispecific antibody that binds to two different regions of LRP6 comprises (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 17; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 18; And (c) a first VH domain comprising at least one, at least two or all three VH HVR sequences selected from HVR-H3 comprising the amino acid sequence of SEQ ID NO: 21; (d) HVR-H1 &lt; / RTI &gt; (e) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 23; And (f) a second VH domain comprising at least one, at least two or all three VH HVR sequences selected from HVR-H3 comprising the amino acid sequence of SEQ ID NO: 24. In one embodiment, the isolated bispecific antibody that binds to two different regions of LRP6 comprises (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 17; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 18; And (c) a first VH domain comprising all three VH HVR sequences from HVR-H3 comprising the amino acid sequence of SEQ ID NO: 21, (d) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 22; (e) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 23; And (f) a second VH domain comprising all three VH HVR sequences from HVR-H3 comprising the amino acid sequence of SEQ ID NO: 24.

In one embodiment, the isolated bispecific antibody that binds to two different regions of LRP6 comprises (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 20; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 18; And (c) a first VH domain comprising at least one, at least two or all three VH HVR sequences selected from HVR-H3 comprising the amino acid sequence of SEQ ID NO: 19, (d) HVR-H1 &lt; / RTI &gt; (e) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 23; And (f) a second VH domain comprising at least one, at least two or all three VH HVR sequences selected from HVR-H3 comprising the amino acid sequence of SEQ ID NO: 24. In one embodiment, the isolated bispecific antibody that binds to two different regions of LRP6 comprises (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 20; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 18; And (c) a first VH domain comprising all three VH HVR sequences from HVR-H3 comprising the amino acid sequence of SEQ ID NO: 19, (d) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 22; (e) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 23; And (f) a second VH domain comprising all three VH HVR sequences from HVR-H3 comprising the amino acid sequence of SEQ ID NO: 24.

In one embodiment, the bispecific antibody of this embodiment comprises: (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 25; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 26; (c) at least one, at least two or all three VL HVR sequences selected from HVR-L3 comprising the amino acid sequence of SEQ ID NO: 27.

In one embodiment, the bispecific antibody of this embodiment comprises: (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 25; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 26; (c) at least one, at least two or all three VL HVR sequences selected from HVR-L3 comprising the amino acid sequence of SEQ ID NO: 28.

One embodiment comprises a first VH comprising the amino acid sequence of SEQ ID NO: 15, and a second VH selected from the group consisting of VH comprising the amino acid sequence of SEQ ID NO: 11 and SEQ ID NO: 13, two different regions of LRP6 RTI ID = 0.0 &gt; bispecific &lt; / RTI &gt; In one embodiment, the antibody further comprises a VL comprising the amino acid sequence of SEQ ID NO: 10 or SEQ ID NO: 12. In one embodiment, the bispecific antibody comprises a first VH comprising the amino acid sequence of SEQ ID NO: 15, a second VH comprising the amino acid sequence of SEQ ID NO: 9, and a VL comprising the amino acid sequence of SEQ ID NO: 10.

In one embodiment, the bispecific antibody inhibits signaling induced by a Wnt isoform selected from the group consisting of Wnt3 and Wnt3a and is selected from the group consisting of Wnt 1, 2, 2b, 6, 8a, 9a, 9b and 10b Inhibits signal transduction induced by selected Wnt isoforms. In one embodiment, the bispecific antibody further inhibits signal transduction induced by a Wnt isoform selected from the group consisting of Wnt 4, 7a, 7b and 10a. In one embodiment, bispecific antibodies inhibit autocrine Wnt signaling.

One aspect of the invention is a method of inhibiting signaling induced by Wnt isoforms selected from the group consisting of Wnt3 and Wnt3a and inhibiting signal transduction induced by Wnt isoforms selected from the group consisting of Wnt 1, 2, 2b, 6, 8a, 9a, 9b and 10b Lt; RTI ID = 0.0 &gt; and / or &lt; / RTI &gt; In one embodiment, the bispecific antibody further inhibits signal transduction induced by Wnt isoforms selected from the group consisting of Wnt 4, 7a, 7b and 10a.

One aspect of the invention provides antibodies that compete for binding to LRP6 with any anti-LRP6 antibody, including bispecific antibodies, as described herein.

Another aspect of the invention provides antibodies that bind to the same two epitopes as the bispecific antibodies described herein. In one embodiment, one of the two epitopes comprises amino acid residues R28, E51, D52, V70, S71, E73, L95, S96, D98, E115, R141 and N185 of LRP6. In one embodiment, one of the two epitopes comprises amino acid residues R28, E51, D52, V70, S71, E73, L95, S96, D98, E115, R141, N185, R29, W188, K202, P225, H226, S243 and F266.

Another aspect of the invention provides an isolated nucleic acid encoding the anti-LRP6 antibody described herein. Another aspect provides a host cell comprising such a nucleic acid.

One aspect of the invention provides an immunoconjugate comprising the anti-LRP6 antibody described herein and a cytotoxic agent. Another aspect provides a pharmaceutical formulation comprising the anti-LRP6 antibody described herein and a pharmaceutically acceptable carrier.

One aspect of the invention provides an anti-LRP6 antibody described herein for use as a medicament. One aspect provides an anti-LRP6 antibody described herein for use in the treatment of cancer or skeletal disorders. One aspect provides the anti-LRP6 antibodies described herein for use in inhibiting signaling induced by a first Wnt isoform and enhancing signaling induced by a second Wnt isoform. One aspect provides for the use of the anti-LRP6 antibodies described herein for use in the manufacture of a medicament useful, for example, in the treatment of cancer or skeletal disorders.

One aspect of the invention is a method of treating an individual, comprising administering to an individual having a cancer, such as non-small cell lung cancer, breast cancer, pancreatic cancer, ovarian cancer, kidney cancer and prostate cancer, an effective amount of an anti- .

1a. A graph showing inhibition of Wnt luciferase reporter activity in HEK293 cells induced with 0.1 mg / ml purified Wnt3a by antibodies against the LRP6.E3-E4 protein.
1b. Western blot analysis of HEK293 cells treated with unlabeled or Wnt3a-induced, indicated LRP6 antibody or purified protein.
1C. A graph showing that the YW210.09 antibody enhances Wnt reporter gene activity in a manner proportional to the Wnt3a concentration in HEK293 cells.
2a. Graph showing the concentration-dependent inhibition and enhancement of self-secreted Wnt signaling in PA-I adenocarcinoma cells transfected with luciferase reporter and either individually or in combination with LRP6 antibody or treated with Fzd8CRD-Fc protein .
2b. 1 cells treated or not treated with 0.3 mg / ml Wnt3a protein and treated with 10 mg / ml YW211.31 antibody, anti-gD monoclonal antibody (negative control) or Fzd8 CRD-Fc protein (positive control) Of the Wnt-induced genes SAX1 and GAD1 and the Wnt-inhibited gene LEFTY2. Data are normalized to samples from cells without (Wnt3a) added (NA).
Figure 3. Summary table showing the effect of LRP6 antibody and Fzd8CRD-Fc protein on autocrine signaling in cell lines.
4a. A graph showing the results of qPCR expression analysis of AXIN2 mRNA in four cell lines treated with 25 [mu] g / ml YW211.31.57 antibody or Fzd8CRD-Fc protein under the presence and absence (NA) of 0.2 [mu] g / ml Wnt3a.
4b. A graph showing that the expression of Wnt-induced genes in NCI-H23 cells is enhanced by YW211.31.57 and antagonized by the YW210.09 antibody (30 [mu] g / ml). CD4-Fc protein (30 μg / ml) serves as a negative control.
4c. A graph showing that the expression of Wnt-induced genes in M14 cells is enhanced by YW211.31.57 and antagonized by the YW210.09 antibody (30 [mu] g / ml).
4d. A graph showing concentration-dependent inhibition of Wnt3a-stimulated signaling by YW211.31.57 antibody in Hs578T cells stably integrated with Wnt luciferase reporter.
4E. A graph showing concentration-dependent enhancement of autocrine Wnt signaling signaling by YW211.31.57 antibody in Hs578T cells stably integrated with Wnt luciferase reporter.
4f. A graph showing that EKVX cells transfected with the Wnt luciferase reporter exhibit potentiation of autocrine Wnt signaling (NA) by the YW211.31.57 antibody and antagonism of Wnt3a-induced signaling.
4g. A graph showing that antibody-mediated enhancement of autocrine Wnt signaling is inhibited by the 5 [mu] g / ml Fzd8CRD-Fc protein.
Figure 5. Effect of 10 mg / ml LRP6 antibody or Fzd8CRD-Fc protein on transfection-induced signal transduction of expression constructs against Wnt isoforms in HEK293 or Hs578T cell lines stably integrated with Wnt luciferase reporter Summary table. Expression of the Wnt luciferase reporter was normalized to the cell number and further normalized to the level in cells that were transfected with the same expression construct but were untreated with protein.
6. A summary table of effects of 10 mg / ml LRP6 antibody or Fzd8CRD-Fc protein on signal transduction in a HEK293 cell line stably integrated with Wnt luciferase reporter. Signal transduction was induced by transfection of expression constructs against chimeric proteins composed of FZD isoforms or Wnt isoforms fused to LRP6. Expression of the Wnt luciferase reporter was normalized to the cell number and further normalized to the level in cells that were transfected with the same expression construct but were untreated with protein.
Figure 7. Summary table of effects of 10 mg / ml LRP6 antibody or antibody combination on transfection-induced signal transduction of expression constructs against Wnt isoforms in a stable integrated cell line with Wnt luciferase reporter. Expression of the Wnt luciferase reporter was normalized to the cell number and further normalized to the level in cells that were transfected with the same expression construct but were untreated with protein.
8a. A graph showing that the combination of YW211.31.57 and YW210.09 antibodies inhibits signal transduction in HEK293 cells stably integrated with Wnt luciferase reporter transfected for expression of both Wnt3a, Wntl, or Wnt3a and Wntl. Anti-gD antibodies and Fzd8CRD-Fc proteins are presented as negative or positive controls, respectively, for inhibition of Wnt signaling.
8b. A graph showing that the combination of YW211.31.57 and YW210.09 antibodies enhances autocrine Wnt signaling in Hs578T cells.
8c. A graph showing that the combination of YW211.31.57 and YW210.09 antibodies enhances autocrine Wnt signaling in EKVX cells.
Figures 9a and b. A biotinylated LRP6 E1-E4 protein immobilized on a streptavidin biosensor, wherein the YW211.31.57 antibody inhibits the binding of Wnt3a and Wnt9b to LRP6 and the YW210.09 antibody inhibits only Wnt9b binding Layer interferometry test.
9c. Bi-layer interferometry assay with smaller non-overlapping fragments of LRP6 showing that Wnt3a binds to the E3-E4 region and that this interaction is blocked by the intact or 1-arm YW211.31 antibody.
9d. Bi-layer interferometry assay with smaller non-overlapping fragments of LRP6 showing that the YW210.09 antibody binds to the LRP6 E1-E2 protein fragment and competes with Wnt9b binding.
9E. A biolayer interferometry assay showing that the YW211.31.57 and YW210.09 antibodies can bind together to a fixed LRP6.E1-E4 protein when sequenced in any order, confirming independent epitopes.
10a. A graph showing MMTV-Wnt1 allograft tumor regression of growth similar to that observed in Fzd8CRD-Fc protein when mice were treated with YW210.09 antibody.
10b. A graph showing that Ntera-2 xenograft tumors show reduced expression of SP5 mRNA by qPCR analysis in mice treated with intact or 1-arm YW211.31 antibody, but not with YW210.09 antibody .
10c. In the cultures, the YW210.09 antibody treatment of the open mouth grafts significantly increased the bone mineral density (BMD) of calcified bony follicles, similar to the treatment with RANK-Fc protein, but not with the YW211.31.62 antibody treatment Graph showing that.
11a. this. The bispecific anti-LRP6 antibody produced in E. coli or HEK293 cells similarly inhibited the Wnt luciferase reporter activity in HEK293 cells induced with 0.1 μg / ml purified Wnt3a in a concentration-dependent manner Graph showing that. The IC 50 values are 0.032 and 0.014 μg / ml, respectively.
11b. Treated with Wnt luciferase (10 μg / ml each), with a control buffer (PBS), antibody, antibody combination or Fzd8CRD-Fc protein (each 10 μg / ml) indicated under presence (c) or absence (b) of stimulation with 0.1 μg / ml Wnt3a (PBS), antibody, antibody combination or Fzd8CRD-Fc protein for self-secreted Wint signaling in CAL-51 cells transfected with stably integrated PA-1 and M14 cells and reporter &lt; / RTI &gt; mg / ml).
11c. (PBS), antibody, antibody combination, or combination of antibodies against CAL-51 cells transfected with PA-1 and M14 cells and reporter cells stably integrated with Wnt luciferase reporter stimulated with 0.1 μg / ml Wnt3a A graph showing the effect of treatment with Fzd8CRD-Fc protein (10 [mu] g / ml each).
Figure 12. Summary of the effect of antibodies or Fzd8CRD protein (10 μg / ml) on the transfection-induced signal transduction of expression constructs against Wnt isoforms in HEK293 or Hs578T cell lines stably integrated with Wnt luciferase reporter table.
13a. Western analysis of HEK293 cells treated with the indicated antibodies or Fzd8CRD-Fc protein (5 [mu] g / ml) for 18 hours in the presence or absence of Wnt3a transfection. beta -actin or GAPDH protein levels are presented as sample loading controls for the top and bottom gels, respectively.
13b. M14 xenograft tumors in SCID-bg mice treated with 30 mg / kg LRP6 bispecific antibody or Fzd8CRD protein for 16 hours exhibited reduced expression of AXIN2 and APCDDl mRNA by qPCR analysis, but with a control anti-gD antibody A graph showing that this is not the case when processed.
Figure 14. Detail view of CDR H3 interaction with residues of LRP6 grooves showing an important network of interactions created by the NAVK motif.
Figure 15. Details of the interactions created by CDR H1,2, L1,2 and 3.
16. The heavy chain variable region (VH) of an exemplary anti-LRP6 antibody showing a Kabat CDR.
17. The light chain variable region (VL) of an exemplary anti-LRP6 antibody showing a Kabat CDR.

Detailed Description of Embodiments of the Present Invention [

I. Definition

For the purposes of this application, " human recipient human framework " means a light chain variable domain (VL) framework or a heavy chain variable domain (VH) framework derived from a human immunoglobulin framework or human consensus framework Is a framework comprising amino acid sequences. An " acceptor human framework ", derived from a human immunoglobulin framework or human consensus framework, may comprise its identical amino acid sequence or may contain amino acid sequence alterations. In some embodiments, the number of amino acid changes is 10 or less, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or less, or 2 or less. In some embodiments, the VL acceptor human framework is identical in sequence to the VL human immunoglobulin framework sequence or human consensus framework sequence.

&Quot; Affinity " refers to the strength of the sum of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., antigen). Unless otherwise indicated, " binding affinity " as used herein refers to an endogenous binding affinity that reflects a 1: 1 interaction between members of a binding pair (e.g., an antibody and an antigen). The affinity of molecule X for partner Y can generally be expressed as the dissociation constant (Kd). The affinity can be measured by conventional methods known in the art including the methods described herein. Specific and exemplary embodiments for binding affinity measurement are described below.

An " affinity matured " antibody refers to an antibody having one or more alterations in one or more hypervariable regions (HVR) relative to a parent antibody that does not have alterations that improve the affinity of the antibody for the antigen.

The terms " anti-LRP6 antibody " and " antibody binding to LRP6 " refer to antibodies capable of binding to LRP6 with sufficient affinity such that the antibody is useful as a diagnostic and / or therapeutic agent in targeting LRP6. In one embodiment, the degree of binding of the anti-LRP6 antibody to an unrelated non-LRP6 protein is less than about 10% of the binding of the antibody to LRP6 as measured by, for example, a radioimmunoassay (RIA) . In certain embodiments, antibodies that bind to LRP6 is ≤ 1 μM, ≤ 100 nM, ≤ 10 nM, ≤ 1 nM, ≤ 0.1 nM, ≤ 0.01 nM , or ≤ 0.001 nM (e.g., 10 -8 M or less, e.g. (10 -8 M to 10 -13 M, for example 10 -9 M to 10 -13 M) dissociation constants (Kd). In certain embodiments, the anti-LRP6 antibody binds to an epitope of LRP6 conserved between LRP6 from different species.

The term " antibody " is used herein in its broadest sense and includes monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e. G., Bispecific antibodies), and antibody fragments that exhibit the desired antigen- But are not limited to, various antibody structures.

&Quot; Antibody fragment " refers to a molecule other than an intact antibody, including a portion of an intact antibody that binds to an antigen to which the intact antibody binds. Examples of antibody fragments include Fv, Fab, Fab ', Fab'-SH, F (ab') 2 ; Diabody; Linear antibodies; Single-chain antibody molecules (e. G., ScFv); And multispecific antibodies formed of antibody fragments.

As " an antibody that binds to the same epitope " as a reference antibody is meant an antibody that blocks 50% or more of the binding of the reference antibody to its antigen in the competition assay, and, conversely, that the reference antibody binds the antigen to its antigen Lt; / RTI &gt; An exemplary competitive assay is provided herein.

The terms " cancer " and " cancerous " typically refer to or describe the physiological condition of a mammal characterized by unregulated cell growth / proliferation. Examples of cancers include, but are not limited to, carcinoma, lymphoma (e. G., Hodgkin and non-Hodgkin's lymphoma), blastoma, sarcoma and leukemia. More specific examples of such cancers are squamous cell carcinoma, small cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, squamous cell carcinoma of the lung, peritoneal cancer, hepatocellular carcinoma, gastric cancer, appendix carcinoma, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer , Liver cancer, bladder cancer, hepatocellular carcinoma, breast cancer, colon cancer, colorectal cancer, endometrial or uterine carcinoma, salivary carcinoma, kidney cancer, liver cancer, prostate cancer, vulvar cancer, thyroid cancer, liver carcinoma, leukemia and other lymphoproliferative disorders, Type head and neck cancer.

&Quot; Chemotherapeutic agent " refers to a chemical compound useful in the treatment of cancer. Examples of chemotherapeutic agents include alkylating agents such as thiotepa and cycloalkyl sports SPA imide (not toksan (CYTOXAN) ®); Alkyl sulphonates such as benzyl sulphate, impro sulphate, and iposulfan; Aziridine, such as benzodopa, carbobucone, metouredopa and ureidopa; Ethyleneimine and methylamelamine (including althretamine, triethylene melamine, triethylenephosphoramide, triethylenethiophosphoramide and trimethylolmelamine); Acetogenin (especially bulatacin and bulatacinone); Delta-9-tetrahydro-compartment butterfly play (draw butterfly play horses play (MARINOL) ®); Beta-rapacon; Rafacall; Colchicine; Betulinic acid; Camptothecin (synthetic analogue topotecan (Hi kamtin (HYCAMTIN) ®), 11-CPT (comprising irinotecan, Kam neoplasm? Sar (CAMPTOSAR), ®), acetyl camptothecin, Scoring pole lectin and 9-amino-camptothecin); Bryostatin; Calistatin; CC-1065 (including its adogelesin, carzelesin and non-gelsin analogs); Grape philatoxin; Grapefinal acid; Tenifocide; Cryptophycin (especially cryptophycin 1 and cryptophycin 8); Dolastatin; Duo &lt; / RTI &gt; carmycine (including synthetic analogues KW-2189 and CB1-TM1); Eleuterobin; Pancreatistin; Sarcocticin; Sponge statin; Nitrogen mustards such as chlorambucil, chlorpavastine, chlorophosphamide, estramustine, ifposamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novolacin, penestherin, fred Nimustine, troposphamide, uracil mustard; Nitrosoureas such as carmustine, chlorochocosin, potemustine, lomustine, nimustine and ranimustine; Antibiotics such as enedin antibiotics (e.g., calicheamicin, especially calicheamicin gamma 1I and calicheamicin omega I1 (see for example Nicolaou et al., Angew. Chem Int. Ed. Engl. 33: 183-186 (1994)); CDP323, oral alpha-4 integrin inhibitor; dynemicin (including dyneimin A); esperamicin; as well as neocarzinostatin chromophore and related chromogenic proteins, Antibiotic chromophore), acclinomycin, actinomycin, auramycin, azaserine, bleomycin, cactinomycin, carabicin, carminomycin, carzinophilin, chromomycin, dactinomycin, daunorubicin, to mound bisin, 6-diazo-5-oxo -L- norleucine, doxorubicin (adriamycin (aDRIAMYCIN) ®, morpholino-doxorubicin, cyano, morpholino-doxorubicin, 2-pyrrolidin-no-doxorubicin, doxorubicin HCl liposome injection (a single room (DOXIL) ®), liposomes Sole bisin TLC D-99 (Mio set (MYOCET) ®), peg screen liposomal doxorubicin containing (kael Riggs (CAELYX) ®) and deoxy doxorubicin), epirubicin, the sole bisin, idarubicin, Marcelo azithromycin, Mito But are not limited to, mucins such as mitomycin C, mycophenolic acid, nogalamycin, olibomycin, pelopromycin, porphyromycin, puromycin, couelramycin, rhodorubicin, streptonigin, streptozocin, Benimex, zinostatin, and zorubicin; Anti-metabolites as, for example, methotrexate, gemcitabine (Gem cut (GEMZAR) ®), Te is greener (Soap toral (UFTORAL) ®), capecitabine (gel loader (XELODA) ®), epothilone, and 5-fluorouracil Uracil (5-FU); Folic acid analogues such as denonfterin, methotrexate, proteopterin, trimethrexate; Purine analogs such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; Pyrimidine analogs such as ancitabine, azacytidine, 6-azauridine, carmopur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, phloxuridine; Androgens such as carrousosterone, dromoglomerolone propionate, epithiostanol, meptiostane, testolactone; Anti-adrenals, such as aminoglutethimide, mitotan, triostane; Folic acid supplements, such as proline acid; Acetic acid; Aldopospermydoglycoside; Aminolevulinic acid; Enyluracil; Amsacrine; Best La Vucil; Arsenate; Edatroxate; Depopamin; Demechecine; Diaziquone; Elformin; Elliptinium acetate; Epothilone; Etoglucide; Gallium nitrate; Hydroxyurea; Lentinan; Ronidainine; Maytansinoids such as maytansine and ansamitocin; Mitoguazone; Mitoxantrone; Fur monotherapy; Nitraerine; Pentostatin; Phenamate; Pyra rubicin; Rosantanone; 2-ethylhydrazide; Procarbazine; PSK ® polysaccharide complex (JHS Natural Products, Eugene, Oreg.); Lauric acid; Liqin; Xanthopyran; Spirogermanium; Tenuazonic acid; Triazicone; 2,2 ', 2'-trichlorotriethylamine; Tricothexene (especially T-2 toxin, veracurin A, loridine A and angiidine); urethane; New binde eldi Shin (ELDISINE) ®, Phil decyne (FILDESIN)); Dakar Basin; Mannostin; Mitobronitol; Mitolactol; Pipobroman; Astaxanthin; Arabinoside (&quot; Ara-C &quot;);Thiotepa; Takso Id, for example, paclitaxel (Taxol (TAXOL) ®); Of paclitaxel albumin-nanoparticle formulation operation (Havre Leshan (ABRAXANE) TM) takso terephthalate (TAXOTERE)), and dock washing cells; Clorane boiling; 6-thioguanine; Mercaptopurine; Methotrexate; Platinum agents such as cisplatin, oxaliplatin (e.g., elrok Satin (ELOXATIN) ®) and carboplatin; Vinblastine (belban (VELBAN) ®), called vincristine, including (on Corbin (ONCOVIN) ®), binde God (eldi New ®, Phil decyne ®), and vinorelbine (or belbin (NAVELBINE) ®), Brno Vinca which prevents polymerization from forming microtubules; Etoposide (VP-16); Iospasmide; Mitoxantrone; Leucovorin; Nobanthrone; Etrexate; Daunomaisin; Aminopterin; Ibandronate; Topoisomerase inhibitors RFS 2000; Difluoromethylornithine (DMFO); Retinoids, such as (X captured vectors (including the tar retinoic (TARGRETIN) ®)) retinoic acid; Bisphosphonates, such as a carbonate (e.g., Bonnet Force (BONEFOS) ® or O Stark (OSTAC) ®), a carbonate (dideuro knife (DIDROCAL) ®), NE- 58095, Sol Red acid / Zoledronic suited to Claude carbonate (crude meth (ZOMETA) ®), alendronate (Fosamax (FOSAMAX) ®), pamidronate (Arenas Dia (AREDIA) ®), Nate as butyl Ruud (skeletal lead (SKELID) ®), or risedronate (aktonel (ACTONEL) ® ); Trocositabine (1,3-dioxolanucleoside cytosine analog); Antisense oligonucleotides, particularly those that inhibit the expression of genes in signal transduction pathways involved in abnormal cell proliferation, such as PKC-alpha, Raf, H-Ras, and epidermal growth factor receptor (EGF-R); Vaccines, such as TB Saratov (THERATOPE) ® vaccine and gene therapy vaccines, for example, egg bektin (ALLOVECTIN) ® vaccine, flow bektin (LEUVECTIN) ® vaccine, and a back oxide (VAXID) ® vaccine; Topoisomerase 1 inhibitor (e. G., Ruhr totekan (LURTOTECAN) ®); rmRH (e.g., O ® barrel Riggs (ABARELIX)); BAY439006 (Sorafanib; Bayer); SU-11248 (sunitinib, can tent (SUTENT) ®, Pfizer (Pfizer)); Peroxisome, a COX-2 inhibitor (e. G., Celecoxib or etoricoxib), a proteosome inhibitor (e. G., PS341); Bortezomib (VELCADE ® ); CCI-779; Tififarinib (R11577); Orapenib, ABT510; dehydrogenase sense (GENASENSE)) Bcl-2 inhibitors, such as Oblique reamer metallocene sodium; Gt; EGFR inhibitors (see below definition); Tyrosine kinase inhibitors (see below); Serine-threonine kinase inhibitors such as rapamycin (sirolimus, Rapa myun (RAPAMUNE) ®); Parnesyl transferase inhibitors such as ronafarnib (SCH 6636, SARASAR TM ); And pharmaceutically acceptable salts, acids or derivatives of any of the foregoing; The only and not the one that two or more of a combination of water, for example, CHOP (cyclophosphamide, doxorubicin, vincristine, and the abbreviation for the combination therapy of prednisolone) and FOLFOX (5-FU and leucovorin combination oxaliplatin (elrok satin TM )). &Lt; / RTI &gt;

Chemotherapeutic agents as defined herein include " antihormonal agents " or " endocrine agents " that act to modulate, reduce, block or inhibit the effects of hormones that can promote the growth of cancer. These wherein with mixed agonist / antagonist profile, estrogen, for example, tamoxifen (nolba index (NOLVADEX) ®), 4- hydroxy tamoxifen, toremifene (Pare stone (FARESTON) ®), idoxifene, deurol hydroxy pen , raloxifene (Avista (EVISTA) ®), tree oxy pen, Kane oxy pen, and selective estrogen receptor modulator (SERM), for example, SERM3; Efficacy of pure anti-estrogens that do not have a second characteristic, for example the pool best sealant (Fossil to Dex (FASLODEX) ®), and EM800 (such agents may block estrogen receptor (ER) dimerization, and / or inhibit the DNA-binding and / or , Increase ER turnover and / or inhibit ER level); Aromatase inhibitors such as steroidal aromatase inhibitors, such as shemesh formate methoxy Stan and exo carbon (aromatic Shin (AROMASIN) ®), and nonsteroidal aromatase inhibitors such as ANA stripe sol (ahrimidekseu (ARIMIDEX) ®), in letrozole (pemara (FEMARA) ®) and amino glue teti imide, and other aromatase inhibitors such as Boro sol (Libby sorbitan (RIVISOR) ®), megestrol acetate (MB three (MEGASE) ®), Pas Sol and 4 (5) -imidazole; Luteinized hormone-releasing hormone agonists, such as leuprolide (loop Ron (LUPRON) ® and Eli guard (ELIGARD) ®, Kose relrin, biasing relrin and trip Te relrin; sex steroids, such as progestins, such as methoxy Estrogen, such as diethylstilbestrol and premarin, and androgens / retinoids, such as, for example, fluoxymasterone, all trans-retinoic acid and fenretinide, onapristone, anti-progesterone, estrogen Receptor antagonists, receptor down-regulators (ERD), antiandrogens such as flutamide, nilutamide and bicalutamide, and pharmaceutically acceptable salts, acids or derivatives of any of the above, as well as two or more of the above But is not limited to, a combination thereof.

The term " chimeric " antibody refers to an antibody in which a portion of the heavy and / or light chain is derived from a particular source or species, while the remainder of the heavy chain and / or light chain is derived from another source or species.

A " class " of an antibody refers to a type of constant domain or constant region retained by its heavy chain. Of five major classes antibodies: IgA, IgD, IgE, IgG and IgM are present, some of which are a subclass (isotype), such as IgG 1, IgG 2, IgG 3, IgG 4, IgA 1, and IgA 2 can be further divided into. The heavy chain constant domains corresponding to the different classes of immunoglobulins are called alpha, delta, epsilon, gamma and mu, respectively.

The term " cytotoxic agent " as used herein refers to a substance that causes inhibition or inhibition of cellular function and / or cell death or destruction. Cytotoxic agents include radioactive isotopes such as At 211 , I 131 , I 125 , Y 90 , Re 186 , Re 188 , Sm 153 , Bi 212 , P 32 , Pb 212 , A chemotherapeutic agent or a drug (e.g., methotrexate, adriamycin, vinca alkaloid (vincristine, vinblastine, etoposide), doxorubicin, melphalan, mitomycin C, chlorambucil, daunorubicin, ); Growth inhibitors; Enzymes and fragments thereof, such as nucleotide degrading enzymes; Antibiotic; Toxins, such as small molecule toxins or enzymatically active toxins of bacterial, fungal, plant or animal origin (including fragments and / or variants thereof); And various antineoplastic or anti-cancer agents disclosed below.

&Quot; Effector function " refers to the biological activity attributed to the Fc region of an antibody, which differs depending on the antibody isotype. Examples of antibody effector functions include C1q binding and complement dependent cytotoxicity (CDC); Fc receptor binding; Antibody-dependent cell-mediated cytotoxicity (ADCC); Phagocytic action; Down regulation of cell surface receptors (e. G., B cell receptors); And B cell activation.

An " effective amount " of an agonist, e. G., A pharmaceutical formulation, refers to an amount effective to achieve the desired treatment or prophylactic result for the required period of time at the requisite dosage.

The term " Fc region " is used herein to define the C-terminal region of an immunoglobulin heavy chain containing at least a portion of a constant region. The term encompasses native sequence Fc regions and variant Fc regions. In one embodiment, the human IgG heavy chain Fc region extends from Cys226, or Pro230, to the carboxyl-terminal end of the heavy chain. However, the C-terminal lysine (Lys447) of the Fc region may or may not be present. Unless otherwise specified herein, the numbering of amino acid residues within the Fc region or constant region is described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. According to the EU numbering system, also referred to as the EU index, as described in the Public Health Service, National Institutes of Health, Bethesda, MD, 1991.

&Quot; Framework " or " FR " refers to variable domain residues other than hypervariable region (HVR) residues. FRs of a variable domain generally consist of four FR domains: FR1, FR2, FR3 and FR4. Thus, HVR and FR sequences generally appear in VH (or VL) in the following order: FR1-H1 (L1) -FR2-H2 (L2) -FR3-H3 (L3) -FR4.

The terms " full-length antibody ", " intact antibody " and " whole antibody " are used interchangeably herein and refer to an antibody having a structure substantially similar to that of the native antibody or having a heavy chain containing the Fc region Quot;

The terms " host cell, " " host cell strain " and " host cell culture " are used interchangeably and refer to a cell into which an exogenous nucleic acid has been introduced, including the progeny of such a cell. Host cells include " transformants " and " transformed cells " which include progeny derived therefrom irrespective of the number of primary transformed cells and subculture. The offspring may not have exactly the same nucleic acid content as the parent cell, but may contain mutations. Mutant descendants having the same function or biological activity screened or selected for the originally transformed cells are included herein.

A "human antibody" is an antibody that has an amino acid sequence corresponding to the amino acid sequence of an antibody produced by a human or human cell, or that is derived from a non-human source using a human antibody repertoire or other human antibody-encoding sequence. Humanized antibodies comprising non-human antigen-binding moieties in this definition of human antibodies are specifically excluded.

The " human consensus framework " is a framework that represents the most common amino acid residues in the selection of human immunoglobulin VL or VH framework sequences. Generally, selection of a human immunoglobulin VL or VH sequence is from a subgroup of variable domain sequences. Generally, subgroups of sequences are described in Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, NIH Publication 91-3242, Bethesda MD (1991), vol. 1-3]. In one embodiment, in the case of VL, the subgroup is subgroup kappa I as in Kabat et al., Supra. In one embodiment, subgroups in the case of VH are subgroup III as in Kabat et al., Supra.

&Quot; Humanized " antibody refers to a chimeric antibody comprising an amino acid residue from a non-human HVR and an amino acid residue from a human FR. In certain embodiments, a humanized antibody will comprise substantially all at least one, typically two, variable domains, wherein all or substantially all of the HVRs (e.g., CDRs) correspond to those of a non- human antibody, and all Or substantially all FRs correspond to those of a human antibody. Humanized antibodies may optionally comprise at least a portion of an antibody constant region derived from a human antibody. An antibody, e. G., A " humanized form, " of a non-human antibody refers to an antibody that has undergone humanization.

As used herein, the term " hypervariable region " or " HVR " refers to each region of an antibody variable domain in which the sequence forms a hypervariable and / or structurally defined loop (" hypervariable loop "). In general, the native four-chain antibody comprises the following six HVRs; Three (H1, H2, H3) in VH and three (L1, L2, L3) in VL. HVRs generally include amino acid residues from hypervariable loops and / or " complementarity determining regions " (CDRs), with the latter having the highest sequence variability and / or associated antigen recognition. Exemplary hypervariable loops are found at amino acid residues 26-32 (L1), 50-52 (L2), 91-96 (L3), 26-32 (H1), 53-55 (H2), and 96-101 Occurs. (Chothia and Lesk, J. Mol. Biol. 196: 901-917 (1987)). Exemplary CDRs (CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2 and CDR-H3) include amino acid residues 24-34 of L1, 50-56 of L2, 89-97 of H3, H1 31-35B of H2, 50-65 of H2, and 95-102 of H3. (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991)). Except for CDR1 in VH, CDRs generally include amino acid residues that form a hypervariable loop. CDRs also include " specificity determining moieties ", or " SDRs, " which are residues that contact the antigen. SDRs are contained within the region of the CDRs called short-CDRs, or a-CDRs. Exemplary a-CDRs (a-CDR-L1, a-CDR-L2, a-CDR-L3, a-CDR- 34, 50-55 of L2, 89-96 of L3, 31-35B of H1, 50-58 of H2, and 95-102 of H3. (See Almagro and Fransson, Front. Biosci. 13: 1619-1633 (2008)). Unless otherwise indicated, the HVR residues and other residues within the variable domain (e. G. FR residues) are numbered according to Kabat et al., Supra.

An " immunoconjugate " is an antibody conjugated to one or more heterologous molecule (s), including but not limited to cytotoxic agents.

An " individual " or " subject " is a mammal. Mammals include rabbits and rodents (e.g., mice and rats), rabbits, rabbits, rabbits, and rabbits, including, but not limited to, livestock (e.g., cows, sheep, cats, dogs and horses), primates (e. G., Human and non-human primates such as monkeys) But is not limited to these. In certain embodiments, the subject or subject is a human.

An " isolated " antibody is isolated from its natural environment components. In some embodiments, the antibody may be conjugated to an antibody, for example, as determined by electrophoresis (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatography (e.g., ion exchange or reverse phase HPLC) Gt; 95% &lt; / RTI &gt; or greater than 99%. For a review of methods of assessing antibody purity, see, for example, Flatman et al., J. Chromatogr. B 848: 79-87 (2007).

An " isolated " nucleic acid refers to a nucleic acid molecule isolated from a component of its natural environment. Isolated nucleic acids include nucleic acid molecules contained in cells that normally contain nucleic acid molecules, but nucleic acid molecules are present at chromosomal locations other than or at a different chromosomal location than their natural chromosomal location.

&Quot; Isolated nucleic acid encoding an anti-LRP6 antibody " refers to a nucleic acid molecule comprising one or more nucleic acid molecules (such as a single vector or a separate vector, such molecule (s) in the vector and at least one position in the host cell) encoding the antibody heavy chain and light chain Including such nucleic acid molecule (s) present.

As used herein, the term " monoclonal antibody " is used to refer to an antibody obtained from a substantially homogeneous population of antibodies, that is, individual antibodies that make up this population are, for example, And / or bind to the same epitope, except for possible mutant antibodies that contain naturally occurring mutations or are produced during the production of monoclonal antibody formulations. In contrast to polyclonal antibody preparations which typically contain different antibodies directed against different crystallizers (epitopes), each monoclonal antibody of the monoclonal antibody preparation is directed against the monoclonal antibody on the antigen. Thus, the expression " monoclonal " refers to the character of an antibody obtained from a substantially homogeneous population of antibodies and should not be interpreted as requiring the manufacture of antibodies by any particular method. For example, a monoclonal antibody used in accordance with the present invention includes a hybridoma method, a recombinant DNA method, a phage-display method, and a method using transgenic animals containing all or part of a human immunoglobulin locus Can be prepared by a variety of techniques, including, but not limited to, the methods described above and other exemplary methods for producing monoclonal antibodies.

&Quot; Naked antibody " refers to a heterologous moiety (e. G., A cytotoxic moiety) or an antibody that is not conjugated to a radioactive label. Naked antibodies can be present in pharmaceutical preparations.

&Quot; Natural antibody " refers to a naturally occurring immunoglobulin molecule having a varying structure. For example, a natural IgG antibody is a heterotetrameric glycoprotein of about 150,000 daltons consisting of two identical light chains and two identical heavy chains that are disulfide-linked. From the N-terminus to the C-terminus, each heavy chain has three constant domains (CH1, CH2 and CH3) following the variable region (VH) (also referred to as the variable heavy chain or heavy chain variable domain). Similarly, from the N-terminus to the C-terminus, each light chain has a constant light chain (CL) domain following the variable region (VL) (also referred to as the variable light chain or light chain variable domain). The light chain of an antibody can be categorized into one of two types, called kappa (kappa) and lambda (lambda), based on the amino acid sequence of its constant domain.

The term " package insert " is intended to encompass a product that is typically included in a commercial package of a therapeutic product, containing information about indications, uses, dosages, administration, combination therapies, contraindications, and / . &Lt; / RTI &gt;

&Quot; Amino acid sequence identity percent (%) " for a reference polypeptide sequence refers to the number of amino acid residues in the reference polypeptide (s), without aligning the sequence and, if necessary, introducing a gap to achieve maximum sequence identity percent, Is defined as the percentage of amino acid residues in the same candidate sequence as the amino acid residue in the sequence. Alignment to determine percent amino acid sequence identity can be accomplished using a variety of methods within the skill in the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software can do. Those skilled in the art will be able to determine the appropriate parameters for the sequence alignment, including any algorithms necessary to achieve maximum alignment for the full length sequence to be compared. However, for purposes of this disclosure,% amino acid sequence identity values are generated using the sequence comparison computer program ALIGN-2. The ALIGN-2 sequence comparison computer program is owned by Genentech, Inc., and the source code is submitted as a user document to the US Copyright Office (Washington, DC 20559) and is copyrighted by US Copyright Registration No. TXU510087 It is registered. The ALIGN-2 program was developed by Genentech, Inc. (South San Francisco, Calif.), Or can be compiled from source code. The ALIGN-2 program must be compiled for use on UNIX operating systems, including Digital UNIX V4.0D. All sequence comparison parameters are set by the ALIGN-2 program and do not change.

In a given amino acid sequence B, in a situation where ALIGN-2 is used for amino acid sequence comparison, the amino acid sequence identity of a given amino acid sequence A to a given amino acid sequence B, or to a given amino acid sequence B (alternatively, B may be described by the given amino acid sequence B, or a given amino acid sequence A that has or has a specified percentage amino acid sequence identity to a given amino acid sequence B) is calculated as follows:

100 x X / Y fraction

Where X is the number of amino acid residues scored in the same match by the program at the time of program alignment of A and B by the sequence alignment program ALIGN-2 and Y is the total number of amino acid residues of B. It will be appreciated that if the length of amino acid sequence A is not equal to the length of amino acid sequence B, then the% amino acid sequence identity of A to B will not be equal to the% amino acid sequence identity of B to A. Unless specifically stated otherwise, all% amino acid sequence identity values used herein are obtained as described in the above paragraph using the ALIGN-2 computer program.

The term " pharmaceutical formulation " refers to a formulation that exists in a form such that the biological activity of the active ingredients contained therein is effective, and that the formulation does not contain additional ingredients that are unacceptable toxicity to the subject to which it is to be administered.

&Quot; Pharmaceutically acceptable carrier " refers to a component in a pharmaceutical formulation other than the active ingredient that is non-toxic to the subject. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers or preservatives.

As used herein, the term " LRP6 " refers to any natural source from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats) LRP6. &Lt; / RTI &gt; The term encompasses &quot; full-length &quot; unprocessed LRP6 as well as any form of LRP6 generated from processing in cells. The term also includes naturally-occurring variants of LRP6, such as splice variants or allelic variants. The amino acid sequence of exemplary human LRP6 is shown in SEQ ID NO: 29. Also, NCBI registration number AAI43726, Strausberg, R. L., et al., Proc. Natl. Acad. Sci. U.S.A. 99: 16899-16903 (2002) (He, X, et al., Development, 131: 1663-1677 (2004); Chen, M., et al., J. Biol. Chem., 284: 35040-35048 2009).

As used herein, " treatment " (and grammatical variants such as " treating " or " treating ") refers to a clinical intervention to alter the natural course of a subject to be treated, For prevention or during the course of a clinical pathological condition. A preferred therapeutic effect is to prevent the occurrence or recurrence of the disease, to improve symptoms, to reduce any direct or indirect pathological consequences of the disease, to prevent metastasis, to decrease the rate of disease progression, to improve or alleviate the disease condition, Including, but not limited to, prognosis. In some embodiments, the antibodies of the invention are used to delay the onset of the disease or to slow the progression of the disease.

The term " variable region " or " variable domain " refers to the domain of an antibody heavy chain or light chain involved in binding an antibody to an antigen. The variable domains of the heavy and light chains of the native antibody (VH and VL, respectively) generally have a similar structure, with each domain comprising four conserved framework regions (FR) and three hypervariable regions (HVR). (See, for example, Kindt et al. Kuby Immunology, 6 th ed., WH Freeman and Co., page 91 (2007)). A single VH or VL domain may be sufficient to confer antigen- have. In addition, antibodies that bind to a particular antigen can be isolated using a VH or VL domain from an antibody that binds to the antigen to screen for a library of complementary VL or VH domains, respectively. See, for example, Portolano et al., J. Immunol. 150: 880-887 (1993); Clarkson et al., Nature 352: 624-628 (1991).

As used herein, the term " vector " refers to a nucleic acid molecule capable of propagating another nucleic acid to which the vector is linked. The term includes not only vectors as self-replicating nucleic acid constructs but also vectors into which the vector is integrated into the genome of the host cell into which it is introduced. A particular vector may direct expression of the nucleic acid to which the vector is operatively linked. Such vectors are referred to herein as " expression vectors ".

II. Composition and method

The present invention provides anti-LRP6 antibodies with unexpected ability to inhibit signaling by some Wnt isoforms and enhance signal transduction by other isoforms. As described in the examples, the two anti-LRP6 antibodies characterized further exhibit mutual activity, one for antibody antagonism and the other for most Wnt. These two antibodies bind to different regions of LRP6 (as do different Wnt isoforms), and inhibition of signal transduction results from blocking Wnt junctions.

Based on the functional interactions with the anti-LRP6 antibodies of the invention, the 14 tested Wnt isoforms can be classified into three classes: Wnt3 and Wnt3a are inhibited by the anti-LRP6 antibody YW211.31 and anti- Enhanced by LRP6 antibody YW210.09; Wnt 1, 2, 2B., 6, 8A, 9A, 9B and 10B are enhanced by anti-LRP6 antibody YW211.31 and are antagonized by anti-LRP6 antibody YW210.09; And Wnt 4, 7A, 7B and 10A were enhanced by anti-LRP6 antibody YW211.31 and not by anti-LRP6 antibody YW210.09 (Fig. 3c). This classification clearly does not correspond to the proposed phylogeny of the Wnt gene even though the Wnt3 / 3a subfamily is the most evolutionarily diverse (Cho et al., 2010). The combination of anti-LRP6 antibodies that inhibit different classes of Wnt isoforms can be used to provide an effective therapy for treating diseases associated with Wnt signaling.

Antibody-mediated dimerization of LRP6 can enhance signal transduction only when the Wnt isoform is also capable of binding to the complex, perhaps by mobilizing FZD. Endogenous autocrine Wnt signaling in different tumor cells can be antagonized or promoted by LRP6 antibodies. The complexity of these secondary receptor-ligand interactions may allow differentiation of signal transduction by Wnt isoforms and may be used with antibodies to differentially manipulate Wnt signaling in specific tissue or disease states.

In some embodiments, the anti-LRP6 antibody may inhibit autocrine, or endogenous, Wnt signaling in some cell types and enhance autocrine signal transduction in other cell types. In some embodiments, the anti-LRP6 antibody mediates dimerization of LRP6 and simultaneously enhances or enhances signal transduction in the presence of Wnt isoforms that bind to LRP6. In some embodiments, the anti-LRP6 antibody enhances Wnt signaling by inhibiting the binding of Wnt antagonists such as DKK isoform and SOST.

Anti-LRP6 antibodies can be used to selectively control the activated or inhibited process by Wnt isoform-induced signaling. This process is, for example, cell proliferation, cell fate determination and self stem cells from different cancer types - including reproduction and developmental processes. Anti-LRP6 is useful, for example, in the treatment of Wnt mediated disorders, such as cancer, and disorders of the bone or skeletal system and vascular disorders. Examples of cancers that can be treated using the anti-LRP6 antibody include small cell lung cancer, non-small cell lung cancer, hepatocellular carcinoma, gastric cancer, pancreatic cancer, ovarian cancer, liver cancer, bladder cancer, hepatocellular carcinoma, breast cancer, colon cancer, colorectal cancer, Uterine carcinoma, salivary carcinoma, kidney cancer (including renal cell carcinoma), liver cancer, and prostate cancer. Examples of skeletal or bone disorders that can be treated using anti-LRP6 antibodies include osteoporosis, osteoarthritis, fractures, and bone lesions. Examples of vascular disorders that can be treated using anti-LRP6 antibodies include, but are not limited to, retinal vascular diseases such as noriosis, osteoporotic-gonadal glioma syndrome (OPPG), familial exudative vitreoretinopathy (FEVR) , Diabetic retinopathy, age-related macular degeneration, retinopathy of prematurity, Cotes &apos; s disease and cottage-like responses, and retinal artery or vein occlusion, and myocardial-related conditions such as myocardial infarction and ischemic heart disease.

Thus, one aspect of the invention provides an antibody that binds LRP6 that inhibits signaling induced by Wnt isoforms and enhances signal transduction induced by another Wnt isoform. In one embodiment, the antibody inhibits signal transduction by Wnt3 and / or Wnt3a. In one embodiment, the antibody enhances signaling by Wnt 1, 2, 2b, 4, 6, 7a, 7b, 8a, 9a, 9b, 10a and / or 10b. In one embodiment, the antibody inhibits signaling by Wnt3 and / or Wnt3a and inhibits signal transduction by Wnt 1, 2, 2b, 4, 6, 7a, 7b, 8a, 9a, 9b, 10a and / Strengthen. In one embodiment, the antibody inhibits signaling by Wnt3 and Wnt3a and enhances signal transduction by Wnt 1, 2, 2b, 4, 6, 7a, 7b, 8a, 9a, 9b, 10a and 10b. In one embodiment, the anti-LRP6 antibody binds to the E3-E4 region (first and second beta-propellers) of LRP6.

In another embodiment, the antibody inhibits signal transduction by Wnt 1, 2, 2b, 6, 8a, 9a, 9b and / or 10b. In one embodiment, the antibody enhances signaling by Wnt3 and / or Wnt3a. In one embodiment, the antibody inhibits signal transduction by Wnt 1, 2, 2b, 6, 8a, 9a, 9b and / or 10b and enhances signaling by Wnt3 and / or Wnt3a. In one embodiment, the antibody inhibits signal transduction by Wnt 1, 2, 2b, 6, 8a, 9a, 9b and / or 10b and enhances signaling by Wnt3 and / or Wnt3a. In one embodiment, the anti-LRP6 antibody binds to the E1-E2 region (third and fourth beta-propellers) of LRP6.

Another aspect of the invention provides multispecific anti-LRP6 antibodies. As shown in the examples, the multispecific antibody, in some embodiments, has the potential to inhibit all three classes of Wnt isoforms. In one embodiment, the anti-LRP6 antibody is a multispecific antibody capable of binding to two or more different regions or epitopes of LRP6. In one embodiment, the multispecific antibody is a bispecific antibody capable of specifically binding to two different regions of LRP6. In one embodiment, the bispecific antibody binds to the EI-E2 region of LRP6 and to the E3-E4 region of LRP6. In one embodiment, the multispecific antibody inhibits signaling induced by a Wnt isoform selected from the group consisting of Wnt3 and Wnt3a and is selected from the group consisting of Wnt 1, 2, 2b, 6, 8a, 9a, 9b and 10b Inhibits signal transduction induced by selected Wnt isoforms. In one embodiment, the multispecific antibody inhibits signaling induced by Wnt isoforms selected from the group consisting of Wnt3 and Wnt3a and comprises a group consisting of Wnt 1, 2, 2b, 6, 8a, 9a, 9b and 10b , And further inhibits signaling induced by Wnt isoforms selected from the group consisting of Wnt 4, 7a, 7b and 10a. In one embodiment, the multispecific antibody inhibits Wnt signaling induced by a combination of Wnt1 and Wnt3a. In one embodiment, the multispecific antibody inhibits autocrine Wnt signaling.

In certain embodiments, the multispecific antibody inhibits signaling induced by a Wnt isoform selected from the group consisting of Wnt3 and Wnt3a and is selected from the group consisting of Wnt 1, 2, 2b, 6, 8a, 9a, 9b and 10b It is a bispecific antibody that inhibits signal transduction induced by selected Wnt isoforms. In certain embodiments, the multispecific antibody inhibits signaling induced by Wnt isoforms selected from the group consisting of Wnt3 and Wnt3a, and is selected from the group consisting of Wnt 1, 2, 2b, 6, 8a, 9a, 9b and 10b , And further inhibits signaling induced by Wnt isoforms selected from the group consisting of Wnt 4, 7a, 7b and 10a. In certain embodiments, the multispecific antibody is a bispecific antibody that inhibits signaling induced by a combination of Wnt1 and Wnt3a. In one embodiment, the multispecific antibody is a bispecific antibody that inhibits signaling induced by a combination of Wntl and Wnt3a more effectively than a combination of monospecific antibodies having the same specificity as a bispecific antibody.

In certain embodiments, the multispecific antibody is a bispecific antibody that more effectively inhibits autocrine Wnt signaling than a combination of monospecific antibodies having the same specificity as the bispecific antibody.

In certain embodiments, the anti-LRP6 antibody or multispecific anti-LRP6 antibody inhibits Wnt signal transduction by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% , Or more. Inhibition of Wnt signaling can be determined using assays known in the art and described herein. For example, inhibition of Wnt signaling can be determined using a Wnt reporter assay, such as the Wnt luciferase reporter assay described in the Examples.

Inhibition of Wnt signaling can also be determined by monitoring the expression of Wnt target genes such as APCDD1, AXIN2, GADl, LEFTY2 and SAXl, as described in the Examples.

In certain embodiments, the anti-LRP6 antibody or multispecific anti-LRP6 antibody inhibits the expression of Wnt target genes such as APCDDl, AXIN2, GADl, LEFTY2 and SAXl by at least about 10%, 20%, 30%, 40% %, 60%, 70%, 80% or 90%. In one embodiment, expression of a Wnt target gene is determined using gene expression assays, such as PCR (including qPCR).

Another aspect of the invention provides antibodies that bind to LRP6 and compete for binding with any of the anti-LRP6 antibodies described herein. Another aspect of the invention provides an antibody that binds to the same epitope on the same LRP6 as any of the anti-LRP6 antibodies described herein.

A. Exemplary Anti-LRP6 Antibodies

One aspect of the invention provides anti-LRP6 antibodies that are monoclonal antibodies, including chimeric, humanized, or human antibodies. In one embodiment, the anti-LRP6 antibody is generated using a phage library. In one embodiment, the anti-LRP6 antibody is an antibody fragment, such as an Fv, Fab, Fab ', scFv, diabody or F (ab') 2 fragment. In another embodiment, the antibody is a full-length antibody, e. G. An intact IgGl antibody, or another antibody class or isotype as defined herein.

In one embodiment, the anti-LRP6 antibody comprises a heavy chain sequence comprising the amino acid sequence of Table 2. In one embodiment, the anti-LRP6 antibody comprises a light chain sequence comprising the amino acid sequence of Table 2. In one embodiment, the anti-LRP6 antibody comprises heavy chain and light chain sequences comprising the amino acid sequences of Table 2.

In one embodiment, the anti-LRP6 antibody comprises a heavy chain sequence comprising the amino acid sequence of SEQ ID NO: 1. In one embodiment, the anti-LRP6 antibody comprises a light chain sequence comprising the amino acid sequence of SEQ ID NO: 2. In one embodiment, the anti-LRP6 antibody comprises a heavy chain sequence comprising the amino acid sequence of SEQ ID NO: 1 and a light chain sequence comprising the amino acid sequence of SEQ ID NO: 2.

In one embodiment, the anti-LRP6 antibody comprises a heavy chain sequence comprising the amino acid sequence of SEQ ID NO: 3. In one embodiment, the anti-LRP6 antibody comprises a light chain sequence comprising the amino acid sequence of SEQ ID NO: 4. In one embodiment, the anti-LRP6 antibody comprises a heavy chain sequence comprising the amino acid sequence of SEQ ID NO: 3 and a light chain sequence comprising the amino acid sequence of SEQ ID NO: 4.

In one embodiment, the anti-LRP6 antibody comprises a heavy chain sequence comprising the amino acid sequence of SEQ ID NO: 5. In one embodiment, the anti-LRP6 antibody comprises a light chain sequence comprising the amino acid sequence of SEQ ID NO: 6. In one embodiment, the anti-LRP6 antibody comprises a heavy chain sequence comprising the amino acid sequence of SEQ ID NO: 5 and a light chain sequence comprising the amino acid sequence of SEQ ID NO: 6.

In one embodiment, the anti-LRP6 antibody comprises a heavy chain sequence comprising the amino acid sequence of SEQ ID NO: 7. In one embodiment, the anti-LRP6 antibody comprises a light chain sequence comprising the amino acid sequence of SEQ ID NO: 8. In one embodiment, the anti-LRP6 antibody comprises a heavy chain sequence comprising the amino acid sequence of SEQ ID NO: 7 and a light chain sequence comprising the amino acid sequence of SEQ ID NO: 8.

In one embodiment, the anti-LRP6 antibody comprises a heavy chain variable domain (VH) from the amino acid sequence of Table 3. In one embodiment, the anti-LRP6 antibody comprises a light chain variable domain (VL) from the amino acid sequence of Table 3. In one embodiment, the anti-LRP6 antibody comprises VH and VL from the amino acid sequences of Table 3.

In one embodiment, the anti-LRP6 antibody comprises a heavy chain variable domain (VH) from the heavy chain of the amino acid sequence of SEQ ID NO: 1. In one embodiment, the anti-LRP6 antibody comprises a light chain variable domain (VL) from the light chain sequence of the amino acid sequence of SEQ ID NO: 2. In one embodiment, the anti-LRP6 antibody comprises a VH from the heavy chain of the amino acid sequence of SEQ ID NO: 1 and a VL from the light chain sequence of the amino acid sequence of SEQ ID NO: 2.

In one embodiment, the anti-LRP6 antibody comprises a heavy chain variable domain (VH) comprising the amino acid sequence of SEQ ID NO: 9. In one embodiment, the anti-LRP6 antibody comprises a light chain variable domain (VL) comprising the amino acid sequence of SEQ ID NO: 10. In one embodiment, the anti-LRP6 antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 9 and a VL comprising the amino acid sequence of SEQ ID NO: 10.

In one embodiment, the anti-LRP6 antibody comprises a heavy chain variable domain (VH) from the heavy chain of the amino acid sequence of SEQ ID NO: 3. In one embodiment, the anti-LRP6 antibody comprises a light chain variable domain (VL) from the light chain sequence of the amino acid sequence of SEQ ID NO: 4. In one embodiment, the anti-LRP6 antibody comprises a VH from the heavy chain of the amino acid sequence of SEQ ID NO: 3 and a VL from the light chain sequence of the amino acid sequence of SEQ ID NO: 4.

In one embodiment, the anti-LRP6 antibody comprises a heavy chain variable domain (VH) comprising the amino acid sequence of SEQ ID NO: 11. In one embodiment, the anti-LRP6 antibody comprises a light chain variable domain (VL) comprising the amino acid sequence of SEQ ID NO: 12. In one embodiment, the anti-LRP6 antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 11 and a VL comprising the amino acid sequence of SEQ ID NO: 12.

In one embodiment, the anti-LRP6 antibody comprises a heavy chain variable domain (VH) from the heavy chain of the amino acid sequence of SEQ ID NO: 5. In one embodiment, the anti-LRP6 antibody comprises a light chain variable domain (VL) from the light chain sequence of the amino acid sequence of SEQ ID NO: 6. In one embodiment, the anti-LRP6 antibody comprises a VH from the heavy chain of the amino acid sequence of SEQ ID NO: 5 and a VL from the light chain sequence of the amino acid sequence of SEQ ID NO: 6.

In one embodiment, the anti-LRP6 antibody comprises a heavy chain variable domain (VH) comprising the amino acid sequence of SEQ ID NO: 13. In one embodiment, the anti-LRP6 antibody comprises a light chain variable domain (VL) comprising the amino acid sequence of SEQ ID NO: 14. In one embodiment, the anti-LRP6 antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 13 and a VL comprising the amino acid sequence of SEQ ID NO: 14.

In one embodiment, the anti-LRP6 antibody comprises a heavy chain variable domain (VH) from the heavy chain of the amino acid sequence of SEQ ID NO: 7. In one embodiment, the anti-LRP6 antibody comprises a light chain variable domain (VL) from the light chain sequence of the amino acid sequence of SEQ ID NO: 8. In one embodiment, the anti-LRP6 antibody comprises a VH from the heavy chain of the amino acid sequence of SEQ ID NO: 7 and a VL from the light chain sequence of the amino acid sequence of SEQ ID NO: 8.

In one embodiment, the anti-LRP6 antibody comprises a heavy chain variable domain (VH) comprising the amino acid sequence of SEQ ID NO: 15. In one embodiment, the anti-LRP6 antibody comprises a light chain variable domain (VL) comprising the amino acid sequence of SEQ ID NO: 16. In one embodiment, the anti-LRP6 antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 15 and a VL comprising the amino acid sequence of SEQ ID NO: 16.

Another aspect of the invention provides multispecific anti-LRP6 antibodies. In one embodiment, the multispecific antibody comprises a heavy chain comprising at least one amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5 or SEQ ID NO: In one embodiment, the multispecific antibody comprises a heavy chain comprising at least two amino acid sequences of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5 or SEQ ID NO: In one embodiment, the multispecific antibody is a bispecific antibody comprising a heavy chain comprising at least one amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5 or SEQ ID NO: In one embodiment, the bispecific antibody comprises a first heavy chain comprising the amino acid sequence of SEQ ID NO: 1 and a second heavy chain comprising the amino acid sequence of SEQ ID NO: 7. In one embodiment, the bispecific antibody comprises a first heavy chain comprising the amino acid sequence of SEQ ID NO: 3 and a second heavy chain comprising the amino acid sequence of SEQ ID NO: 7. In one embodiment, the bispecific antibody comprises a first heavy chain comprising the amino acid sequence of SEQ ID NO: 5 and a second heavy chain comprising the amino acid sequence of SEQ ID NO: 7.

In one embodiment, the multispecific anti-LRP6 antibody comprises a VH from the heavy chain of at least one amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5 or SEQ ID NO: In one embodiment, the multispecific antibody comprises a VH from the heavy chain of at least two amino acid sequences of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5 or SEQ ID NO: In one embodiment, the multispecific antibody is a bispecific antibody comprising a VH from the heavy chain of the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5 or SEQ ID NO: In one embodiment, the bispecific antibody comprises a VH from the heavy chain of the amino acid sequence of SEQ ID NO: 1 and a VH from the heavy chain of the amino acid sequence of SEQ ID NO: 7. In one embodiment, the bispecific antibody comprises a VH from the heavy chain of the amino acid sequence of SEQ ID NO: 3 and a VH from the heavy chain of the amino acid sequence of SEQ ID NO: 7. In one embodiment, the bispecific antibody comprises a VH from the heavy chain of the amino acid sequence of SEQ ID NO: 5 and a VH from the heavy chain of the amino acid sequence of SEQ ID NO: 7.

In one embodiment, the multispecific anti-LRP6 antibody comprises a VH comprising at least one amino acid sequence of SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13 or SEQ ID NO: In one embodiment, the multispecific antibody comprises a VH comprising at least two amino acid sequences of SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13 or SEQ ID NO: In one embodiment, the multispecific antibody is a bispecific antibody comprising a VH comprising the amino acid sequence of SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13 or SEQ ID NO: In one embodiment, the bispecific antibody comprises a first VH comprising the amino acid sequence of SEQ ID NO: 9 and a second VH comprising the amino acid sequence of SEQ ID NO: 15. In one embodiment, the bispecific antibody comprises a first VH comprising the amino acid sequence of SEQ ID NO: 11 and a second VH comprising the amino acid sequence of SEQ ID NO: 15. In one embodiment, the bispecific antibody comprises a first VH comprising the amino acid sequence of SEQ ID NO: 13 and a second VH comprising the amino acid sequence of SEQ ID NO: 15.

In one embodiment, the anti-LRP6 antibody comprises (a) HVR-H1 from the HVR-H1 amino acid sequence of Table 4; (b) HVR-H2 from the HVR-H2 amino acid sequence of Table 4; (c) HVR-H3 from the HVR-H3 amino acid sequence of Table 4; (d) HVR-L1 from the HVR-L1 amino acid sequence of Table 4; (e) HVR-L2 from the HVR-L2 amino acid sequence of Table 4; And (f) at least 1, 2, 3, 4, 5 or 6 HVRs selected from HVR-L3 from the HVR-L3 amino acid sequence of Table 4.

In one embodiment, the anti-LRP6 antibody comprises (a) a heavy chain HVR-H1 of SEQ ID NO: 1; (b) HVR-H2 of the heavy chain of SEQ ID NO: 1; (c) HVR-H3 of the heavy chain of SEQ ID NO: 1; (d) HVR-L1 of light chain of SEQ ID NO: 2; (e) HVR-L2 of light chain of SEQ ID NO: 2; And (f) a VH comprising at least 1, 2, 3, 4, 5 or 6 HVRs selected from the light chain HVR-L3 of SEQ ID NO: 2.

In one embodiment, the anti-LRP6 antibody comprises (a) a heavy chain HVR-H1 of SEQ ID NO: 1; (b) HVR-H2 of the heavy chain of SEQ ID NO: 1; And (c) at least one, at least two or all three VH HVR sequences selected from the heavy chain HVR-H3 of SEQ ID NO: 1. In one embodiment, the antibody comprises the heavy chain HVR-H3 of SEQ ID NO: 1. In another embodiment, the antibody comprises HVR-H3 of the heavy chain of SEQ ID NO: 1 and HVR-L3 of the light chain of SEQ ID NO: 2. In a further embodiment, the antibody comprises HVR-H3 of the heavy chain of SEQ ID NO: 1, HVR-L3 of the light chain of SEQ ID NO: 2, and HVR-H2 of the heavy chain of SEQ ID NO: In a further embodiment, the antibody comprises (a) a heavy chain HVR-H1 of SEQ ID NO: 1; (b) HVR-H2 of the heavy chain of SEQ ID NO: 1; And (c) the heavy chain HVR-H3 of SEQ ID NO: 1.

In one embodiment, the anti-LRP6 antibody comprises (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 17; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 18; And (c) at least one, at least two or all three VH HVR sequences selected from HVR-H3 comprising the amino acid sequence of SEQ ID NO: 19. In one embodiment, the antibody comprises HVR-H3 comprising the amino acid sequence of SEQ ID NO: 19. In another embodiment, the antibody comprises HVR-H3 comprising the amino acid sequence of SEQ ID NO: 19 and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 27. In a further embodiment, the antibody comprises HVR-H3 comprising the amino acid sequence of SEQ ID NO: 19, HVR-L3 comprising the amino acid sequence of SEQ ID NO: 27, and HVR-H2 comprising the amino acid sequence of SEQ ID NO: 18. In a further embodiment, the antibody comprises: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 17; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 18; And (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 19.

In one embodiment, the anti-LRP6 antibody comprises (a) a light chain HVR-L1 of SEQ ID NO: 2; (b) HVR-L2 of light chain of SEQ ID NO: 2; And (c) at least one, at least two, or all three VL HVR sequences selected from the light chain HVR-L3 of SEQ ID NO: 2. In one embodiment, the antibody comprises (a) a light chain HVR-L1 of SEQ ID NO: 2; (b) HVR-L2 of light chain of SEQ ID NO: 2; And (c) the light chain HVR-L3 of SEQ ID NO: 2.

(Ii) the heavy chain HVR-H2 of SEQ ID NO: 1 and (iii) the heavy chain HVR-H3 of the heavy chain of SEQ ID NO: 1. In one embodiment, the anti-LRP6 antibody comprises A VH domain comprising at least one, at least two, or all three VH HVR sequences; And (b) at least one, at least two, or all selected from (i) HVR-L1 of light chain of SEQ ID NO: 2, (ii) HVR-L2 of light chain of SEQ ID NO: 2, And a VL domain comprising three VL HVR sequences.

(Ii) an HVR-H2 comprising the amino acid sequence of SEQ ID NO: 18 and (iii) an amino acid sequence of SEQ ID NO: 19, wherein the amino acid sequence of SEQ ID NO: A VH domain comprising at least one, at least two or all three VH HVR sequences selected from HVR-H3 comprising a sequence; (Ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 26; and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 27. At least one, at least two, or all three VL HVR sequences.

In another aspect, the invention provides a kit comprising: (a) a heavy chain HVR-H1 of SEQ ID NO: 1; (b) HVR-H2 of the heavy chain of SEQ ID NO: 1; (c) HVR-H3 of the heavy chain of SEQ ID NO: 1; (d) HVR-L1 of light chain of SEQ ID NO: 2; (e) HVR-L2 of light chain of SEQ ID NO: 2; And (f) a light chain HVR-L3 of SEQ ID NO: 2.

In another aspect, the invention provides a kit comprising: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 17; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 18; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 19; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 25; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 26; And (f) an HVR-L3 comprising the amino acid sequence of SEQ ID NO: 27.

In one embodiment, the anti-LRP6 antibody comprises (a) HVR-H1 of the heavy chain of SEQ ID NO: 3; (b) HVR-H2 of the heavy chain of SEQ ID NO: 3; (c) HVR-H3 of the heavy chain of SEQ ID NO: 3; (d) HVR-L1 of light chain of SEQ ID NO: 4; (e) HVR-L2 of light chain of SEQ ID NO: 4; And (f) at least 1, 2, 3, 4, 5 or 6 HVRs selected from the light chain HVR-L3 of SEQ ID NO: 4.

In one embodiment, the anti-LRP6 antibody comprises (a) HVR-H1 of the heavy chain of SEQ ID NO: 3; (b) HVR-H2 of the heavy chain of SEQ ID NO: 3; And (c) at least one, at least two or all three VH HVR sequences selected from the heavy chain HVR-H3 of SEQ ID NO: 3. In one embodiment, the antibody comprises the heavy chain HVR-H3 of SEQ ID NO: 3. In another embodiment, the antibody comprises the heavy chain HVR-H3 of SEQ ID NO: 3 and the light chain HVR-L3 of SEQ ID NO: 4. In a further embodiment, the antibody comprises HVR-H3 of the heavy chain of SEQ ID NO: 3, HVR-L3 of the light chain of SEQ ID NO: 4, and HVR-H2 of the heavy chain of SEQ ID NO: In a further embodiment, the antibody comprises (a) HVR-H1 of the heavy chain of SEQ ID NO: 3; (b) HVR-H2 of the heavy chain of SEQ ID NO: 3; And (c) the heavy chain HVR-H3 of SEQ ID NO: 3.

In one embodiment, the anti-LRP6 antibody comprises (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 17; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 18; And (c) at least one, at least two or all three VH HVR sequences selected from HVR-H3 comprising the amino acid sequence of SEQ ID NO: 21. In one embodiment, the antibody comprises HVR-H3 comprising the amino acid sequence of SEQ ID NO: 21. In another embodiment, the antibody comprises HVR-H3 of the heavy chain of SEQ ID NO: 21 and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 28. In a further embodiment, the antibody comprises HVR-H3 comprising the amino acid sequence of SEQ ID NO: 21, HVR-L3 comprising the amino acid sequence of SEQ ID NO: 28, and HVR-H2 comprising the amino acid sequence of SEQ ID NO: 18. In a further embodiment, the antibody comprises: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 17; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 18; And (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 21.

In one embodiment, the anti-LRP6 antibody comprises (a) a light chain HVR-L1 of SEQ ID NO: 4; (b) HVR-L2 of light chain of SEQ ID NO: 4; And (c) at least one, at least two or all three VL HVR sequences selected from the light chain HVR-L3 of SEQ ID NO: 4. In one embodiment, the antibody comprises (a) a light chain HVR-L1 of SEQ ID NO: 4; (b) HVR-L2 of light chain of SEQ ID NO: 4; And (c) the light chain HVR-L3 of SEQ ID NO: 4.

In one embodiment, the anti-LRP6 antibody comprises (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 25; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 26; And (c) at least one, at least two or all three VL HVR sequences selected from HVR-L3 comprising the amino acid sequence of SEQ ID NO: 28. In one embodiment, the antibody comprises (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 25; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 26; And (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 28.

(Ii) the heavy chain HVR-H2 of SEQ ID NO: 3 and (iii) the heavy chain HVR-H3 of the heavy chain of SEQ ID NO: 3. In one embodiment, the anti-LRP6 antibody comprises A VH domain comprising at least one, at least two, or all three VH HVR sequences; And (b) at least one, at least two or all of (i) the light chain HVR-L1 of SEQ ID NO: 4, (ii) the light chain HVR- And a VL domain comprising three VL HVR sequences.

(Ii) an HVR-H2 comprising the amino acid sequence of SEQ ID NO: 18; and (iii) an amino acid sequence of SEQ ID NO: 21, wherein the amino acid sequence of SEQ ID NO: A VH domain comprising at least one, at least two or all three VH HVR sequences selected from HVR-H3 comprising a sequence; (Ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 26; and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 28. At least one, at least two, or all three VL HVR sequences.

In another aspect, the invention provides a kit comprising (a) a heavy chain HVR-H1 of SEQ ID NO: 3; (b) HVR-H2 of the heavy chain of SEQ ID NO: 3; (c) HVR-H3 of the heavy chain of SEQ ID NO: 3; (d) HVR-L1 of light chain of SEQ ID NO: 4; (e) HVR-L2 of light chain of SEQ ID NO: 4; And (f) an HVR-L3 light chain of SEQ ID NO: 4.

In another aspect, the invention provides a kit comprising: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 17; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 18; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 21; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 25; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 26; And (f) an HVR-L3 comprising the amino acid sequence of SEQ ID NO: 28.

In one embodiment, the anti-LRP6 antibody comprises (a) a heavy chain HVR-H1 of SEQ ID NO: 5; (b) HVR-H2 of the heavy chain of SEQ ID NO: 5; (c) HVR-H3 of the heavy chain of SEQ ID NO: 5; (d) HVR-L1 of light chain of SEQ ID NO: 6; (e) HVR-L2 of light chain of SEQ ID NO: 6; And (f) at least 1, 2, 3, 4, 5 or 6 HVRs selected from the light chain HVR-L3 of SEQ ID NO: 6.

In one embodiment, the anti-LRP6 antibody comprises (a) a heavy chain HVR-H1 of SEQ ID NO: 5; (b) HVR-H2 of the heavy chain of SEQ ID NO: 5; And (c) at least one, at least two or all three VH HVR sequences selected from the heavy chain HVR-H3 of SEQ ID NO: 5. In one embodiment, the antibody comprises the heavy chain HVR-H3 of SEQ ID NO: 5. In another embodiment, the antibody comprises the heavy chain HVR-H3 of SEQ ID NO: 5 and the light chain HVR-L3 of SEQ ID NO: 6. In a further embodiment, the antibody comprises HVR-H3 of the heavy chain of SEQ ID NO: 5, HVR-L3 of the light chain of SEQ ID NO: 6, and HVR-H2 of the heavy chain of SEQ ID NO: In a further embodiment, the antibody comprises (a) HVR-H1 of the heavy chain of SEQ ID NO: 5; (b) HVR-H2 of the heavy chain of SEQ ID NO: 5; And (c) the heavy chain HVR-H3 of SEQ ID NO: 5.

In one embodiment, the anti-LRP6 antibody comprises (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 20; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 18; And (c) at least one, at least two or all three VH HVR sequences selected from HVR-H3 comprising the amino acid sequence of SEQ ID NO: 19. In one embodiment, the antibody comprises HVR-H3 comprising the amino acid sequence of SEQ ID NO: 19. In another embodiment, the antibody comprises HVR-H3 comprising the amino acid sequence of SEQ ID NO: 19 and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 27. In a further embodiment, the antibody comprises HVR-H3 comprising the amino acid sequence of SEQ ID NO: 19, HVR-L3 comprising the amino acid sequence of SEQ ID NO: 27, and HVR-H2 comprising the amino acid sequence of SEQ ID NO: 18. In a further embodiment, the antibody comprises: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 20; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 18; And (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 19.

In one embodiment, the anti-LRP6 antibody comprises (a) a light chain HVR-L1 of SEQ ID NO: 6; (b) HVR-L2 of light chain of SEQ ID NO: 6; And (c) at least one, at least two, or all three VL HVR sequences selected from the light chain HVR-L3 of SEQ ID NO: 6. In one embodiment, the antibody comprises (a) a light chain HVR-L1 of SEQ ID NO: 6; (b) HVR-L2 of light chain of SEQ ID NO: 6; And (c) the light chain HVR-L3 of SEQ ID NO: 6.

(Ii) the heavy chain HVR-H2 of SEQ ID NO: 5 and (iii) the heavy chain HVR-H3 of the heavy chain of SEQ ID NO: 5. In one embodiment, the anti-LRP6 antibody comprises A VH domain comprising at least one, at least two, or all three VH HVR sequences; And (b) at least one, at least two or all three selected from (i) HVR-L1 of the light chain of SEQ ID NO: 6, (ii) HVR-L2 of the light chain of SEQ ID NO: RTI ID = 0.0 &gt; VL &lt; / RTI &gt; HVR sequences.

(Ii) an HVR-H2 comprising the amino acid sequence of SEQ ID NO: 18 and (iii) an amino acid sequence of SEQ ID NO: 19, wherein the amino acid sequence of SEQ ID NO: A VH domain comprising at least one, at least two or all three VH HVR sequences selected from HVR-H3 comprising a sequence; (Ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 26; and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 27. At least one, at least two, or all three VL HVR sequences.

In another aspect, the invention provides a kit comprising: (a) a heavy chain of HVR-H1 of SEQ ID NO: 5; (b) HVR-H2 of the heavy chain of SEQ ID NO: 5; (c) HVR-H3 of the heavy chain of SEQ ID NO: 5; (d) HVR-L1 of light chain of SEQ ID NO: 6; (e) HVR-L2 of light chain of SEQ ID NO: 6; And (f) a light chain HVR-L3 of SEQ ID NO: 6.

In another aspect, the invention provides a kit comprising: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 20; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 18; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 19; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 25; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 26; And (f) an HVR-L3 comprising the amino acid sequence of SEQ ID NO: 27.

In one embodiment, the anti-LRP6 antibody comprises (a) HVR-H1 of the heavy chain of SEQ ID NO: 7; (b) HVR-H2 of the heavy chain of SEQ ID NO: 7; (c) HVR-H3 of the heavy chain of SEQ ID NO: 7; (d) HVR-L1 of light chain of SEQ ID NO: 8; (e) HVR-L2 of light chain of SEQ ID NO: 8; And (f) at least 1, 2, 3, 4, 5 or 6 HVRs selected from the light chain HVR-L3 of SEQ ID NO: 8.

In one embodiment, the anti-LRP6 antibody comprises (a) HVR-H1 of the heavy chain of SEQ ID NO: 7; (b) HVR-H2 of the heavy chain of SEQ ID NO: 7; And (c) at least one, at least two, or all three VH HVR sequences selected from the heavy chain HVR-H3 of SEQ ID NO: 7. In one embodiment, the antibody comprises the heavy chain HVR-H3 of SEQ ID NO: 7. In another embodiment, the antibody comprises the heavy chain HVR-H3 of SEQ ID NO: 7 and the light chain HVR-L3 of SEQ ID NO: 8. In a further embodiment, the antibody comprises HVR-H3 of the heavy chain of SEQ ID NO: 7, HVR-L3 of the light chain of SEQ ID NO: 8, and HVR-H2 of the heavy chain of SEQ ID NO: In a further embodiment, the antibody comprises (a) a heavy chain HVR-H1 of SEQ ID NO: 7; (b) HVR-H2 of the heavy chain of SEQ ID NO: 7; And (c) the heavy chain HVR-H3 of SEQ ID NO: 7.

In one embodiment, the anti-LRP6 antibody comprises (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 22; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 23; And (c) at least one, at least two or all three VH HVR sequences selected from HVR-H3 comprising the amino acid sequence of SEQ ID NO: 24. In one embodiment, the antibody comprises HVR-H3 comprising the amino acid sequence of SEQ ID NO: 24. In another embodiment, the antibody comprises HVR-H3 comprising the amino acid sequence of SEQ ID NO: 24 and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 27. In a further embodiment, the antibody comprises HVR-H3 comprising the amino acid sequence of SEQ ID NO: 24, HVR-L3 comprising the amino acid sequence of SEQ ID NO: 27, and HVR-H2 comprising the amino acid sequence of SEQ ID NO: In a further embodiment, the antibody comprises: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 22; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 23; And (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 24.

In one embodiment, the anti-LRP6 antibody comprises (a) a light chain HVR-L1 of SEQ ID NO: 8; (b) HVR-L2 of light chain of SEQ ID NO: 8; And (c) at least one, at least two or all three VL HVR sequences selected from the light chain HVR-L3 of SEQ ID NO: 8. In one embodiment, the antibody comprises (a) a light chain HVR-L1 of SEQ ID NO: 8; (b) HVR-L2 of light chain of SEQ ID NO: 8; And (c) the light chain HVR-L3 of SEQ ID NO: 8.

(Ii) the heavy chain HVR-H2 of SEQ ID NO: 7 and (iii) the heavy chain HVR-H3 of SEQ ID NO: 7. In one embodiment, the anti-LRP6 antibody comprises A VH domain comprising at least one, at least two, or all three VH HVR sequences; And (b) at least one, at least two or all of the light chain HVR-L1 selected from (i) the light chain HVR-L1 of SEQ ID NO: 8, (ii) the light chain HVR- And a VL domain comprising three VL HVR sequences.

(Ii) an HVR-H2 comprising the amino acid sequence of SEQ ID NO: 23; and (iii) an amino acid sequence of SEQ ID NO: 24, wherein the amino acid sequence of SEQ ID NO: A VH domain comprising at least one, at least two or all three VH HVR sequences selected from HVR-H3 comprising a sequence; (Ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 26; and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 27. At least one, at least two, or all three VL HVR sequences.

In another embodiment, the invention provides a kit comprising: (a) a heavy chain HVR-H1 of SEQ ID NO: 7; (b) HVR-H2 of the heavy chain of SEQ ID NO: 7; (c) HVR-H3 of the heavy chain of SEQ ID NO: 7; (d) HVR-L1 of light chain of SEQ ID NO: 8; (e) HVR-L2 of light chain of SEQ ID NO: 8; And (f) a light chain HVR-L3 of SEQ ID NO: 8.

In another embodiment, the invention provides a kit comprising: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 22; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 23; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 24; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 25; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 26; And (f) an HVR-L3 comprising the amino acid sequence of SEQ ID NO: 27.

In one embodiment, the anti-LRP6 antibody comprises (a) a heavy chain HVR-H1 of SEQ ID NO: 1; (b) HVR-H2 of the heavy chain of SEQ ID NO: 1; And (c) a first VH domain comprising at least one, at least two or all three VH HVR sequences selected from HVR-H3 of the heavy chain of SEQ ID NO: 1, and (d) HVR-H1 of the heavy chain of SEQ ID NO: 7; (e) HVR-H2 of the heavy chain of SEQ ID NO: 7; And (f) a second VH domain comprising at least one, at least two or all three VH HVR sequences selected from the heavy chain HVR-H3 of SEQ ID NO: 7.

In one embodiment, the anti-LRP6 antibody comprises (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 17; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 18; And (c) a first VH domain comprising at least one, at least two or all three VH HVR sequences selected from HVR-H3 comprising the amino acid sequence of SEQ ID NO: 19, and (d) the amino acid sequence of SEQ ID NO: HVR-H1; (e) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 23; And (f) a second VH domain comprising at least one, at least two or all three VH HVR sequences selected from HVR-H3 comprising the amino acid sequence of SEQ ID NO: 24.

In one embodiment, the anti-LRP6 antibody comprises (a) a heavy chain HVR-H1 of SEQ ID NO: 1; (b) HVR-H2 of the heavy chain of SEQ ID NO: 1; And (c) a first VH domain comprising all three VH HVR sequences from HVR-H3 of the heavy chain of SEQ ID NO: 1, and (d) HVR-H1 of the heavy chain of SEQ ID NO: 7; (e) HVR-H2 of the heavy chain of SEQ ID NO: 7; And (f) a second VH domain comprising all three VH HVR sequences from HVR-H3 of the heavy chain of SEQ ID NO: 7.

In one embodiment, the anti-LRP6 antibody comprises (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 17; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 18; And (c) a first VH domain comprising all three VH HVR sequences from HVR-H3 comprising the amino acid sequence of SEQ ID NO: 19, (d) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 22; (e) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 23; And (f) a second VH domain comprising all three VH HVR sequences from HVR-H3 comprising the amino acid sequence of SEQ ID NO: 24.

In one embodiment, the anti-LRP6 antibody comprises (a) HVR-H1 of the heavy chain of SEQ ID NO: 3; (b) HVR-H2 of the heavy chain of SEQ ID NO: 3; And (c) a first VH domain comprising at least one, at least two or all three VH HVR sequences selected from the heavy chain HVR-H3 of SEQ ID NO: 3, (d) a heavy chain HVR- ; (e) HVR-H2 of the heavy chain of SEQ ID NO: 7; And (f) a second VH domain comprising at least one, at least two or all three VH HVR sequences selected from the heavy chain HVR-H3 of SEQ ID NO: 7.

In one embodiment, the anti-LRP6 antibody comprises (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 17; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 18; And (c) a first VH domain comprising at least one, at least two or all three VH HVR sequences selected from HVR-H3 comprising the amino acid sequence of SEQ ID NO: 21; (d) HVR-H1 &lt; / RTI &gt; (e) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 23; And (f) a second VH domain comprising at least one, at least two or all three VH HVR sequences selected from HVR-H3 comprising the amino acid sequence of SEQ ID NO: 24.

In one embodiment, the anti-LRP6 antibody comprises (a) HVR-H1 of the heavy chain of SEQ ID NO: 3; (b) HVR-H2 of the heavy chain of SEQ ID NO: 3; And (c) a first VH domain comprising all three VH HVR sequences from HVR-H3 of the heavy chain of SEQ ID NO: 3, and (d) HVR-H1 of the heavy chain of SEQ ID NO: 7; (e) HVR-H2 of the heavy chain of SEQ ID NO: 7; And (f) a second VH domain comprising all three VH HVR sequences from HVR-H3 of the heavy chain of SEQ ID NO: 7.

In one embodiment, the anti-LRP6 antibody comprises (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 17; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 18; And (c) a first VH domain comprising all three VH HVR sequences from HVR-H3 comprising the amino acid sequence of SEQ ID NO: 21, (d) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 22; (e) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 23; And (f) a second VH domain comprising all three VH HVR sequences from HVR-H3 comprising the amino acid sequence of SEQ ID NO: 24.

In one embodiment, the anti-LRP6 antibody comprises (a) a heavy chain HVR-H1 of SEQ ID NO: 5; (b) HVR-H2 of the heavy chain of SEQ ID NO: 5; And (c) a first VH domain comprising at least one, at least two or all three VH HVR sequences selected from the heavy chain HVR-H3 of SEQ ID NO: 5, (d) ; (e) HVR-H2 of the heavy chain of SEQ ID NO: 7; And (f) a second VH domain comprising at least one, at least two or all three VH HVR sequences selected from the heavy chain HVR-H3 of SEQ ID NO: 7.

In one embodiment, the anti-LRP6 antibody comprises (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 20; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 18; And (c) a first VH domain comprising at least one, at least two or all three VH HVR sequences selected from HVR-H3 comprising the amino acid sequence of SEQ ID NO: 19, (d) HVR-H1 &lt; / RTI &gt; (e) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 23; And (f) a second VH domain comprising at least one, at least two or all three VH HVR sequences selected from HVR-H3 comprising the amino acid sequence of SEQ ID NO: 24.

In one embodiment, the anti-LRP6 antibody comprises (a) a heavy chain HVR-H1 of SEQ ID NO: 5; (b) HVR-H2 of the heavy chain of SEQ ID NO: 5; And (c) a first VH domain comprising all three VH HVR sequences from HVR-H3 of the heavy chain of SEQ ID NO: 5, and (d) HVR-H1 of the heavy chain of SEQ ID NO: 7; (e) HVR-H2 of the heavy chain of SEQ ID NO: 7; And (f) a second VH domain comprising all three VH HVR sequences from HVR-H3 of the heavy chain of SEQ ID NO: 7.

In one embodiment, the anti-LRP6 antibody comprises (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 20; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 18; And (c) a first VH domain comprising all three VH HVR sequences from HVR-H3 comprising the amino acid sequence of SEQ ID NO: 19, (d) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 22; (e) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 23; And (f) a second VH domain comprising all three VH HVR sequences from HVR-H3 comprising the amino acid sequence of SEQ ID NO: 24.

In any of the above embodiments of the multispecific anti-LRP6 antibody, the antibody comprises (a) HVR-L1 of the light chain of SEQ ID NO: 2; (b) HVR-L2 of light chain of SEQ ID NO: 2; And (c) at least one, at least two or all three VL HVR sequences selected from the light chain HVR-L3 of SEQ ID NO: 2 or SEQ ID NO: 4. In one embodiment, the antibody comprises (a) a light chain HVR-L1 of SEQ ID NO: 2; (b) HVR-L2 of light chain of SEQ ID NO: 2; And (c) the light chain HVR-L3 of SEQ ID NO: 2. In one embodiment, the antibody comprises (a) a light chain HVR-L1 of SEQ ID NO: 2; (b) HVR-L2 of light chain of SEQ ID NO: 2; And (c) the light chain HVR-L3 of SEQ ID NO: 4.

In any of the above embodiments of the multispecific anti-LRP6 antibody, the antibody comprises (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 25; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 26; And (c) at least one, at least two or all three VL HVR sequences selected from HVR-L3 comprising the amino acids of SEQ ID NO: 27 or SEQ ID NO: 28. In one embodiment, the antibody comprises (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 25; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 26; And (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 27. In one embodiment, the antibody comprises (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 25; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 26; And (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 28.

In one embodiment, the anti-LRP6 antibody or multispecific anti-LRP6 antibody comprises HVR-H3 comprising the amino acid sequence NX 1 X 2 K (SEQ ID NO: 41). In one embodiment, the anti-LRP6 antibody or multispecific anti-LRP6 antibody comprises HVR-H3 comprising the amino acid sequence NX 1 X 2 KN (SEQ ID NO: 42). In one embodiment, the anti-LRP6 antibody or multispecific anti-LRP6 antibody comprises HVR-H3 comprising the amino acid sequence NX 1 VK (SEQ ID NO: 43). In one embodiment, the anti-LRP6 antibody or multispecific anti-LRP6 antibody comprises HVR-H3 comprising the amino acid sequence NX 1 IK (SEQ ID NO: 44). In one embodiment, the anti-LRP6 antibody or multispecific anti-LRP6 antibody comprises HVR-H3 comprising the amino acid sequence NX 1 VKN (SEQ ID NO: 45). In one embodiment, the anti-LRP6 antibody or the multispecific anti-LRP6 antibody comprises HVR-H3 comprising the amino acid sequence NX 1 IKN (SEQ ID NO: 46). In this embodiment, X 1 is an amino acid and X 2 is I or V; Or X 1 is A, S, F, T, Y or L and X 2 is I or V; Or X 1 is A, S, F, T, Y or L and X 2 is I; Or X 1 is A, S, F, T, Y or L and X 2 is V.

In one embodiment, the anti-LRP6 antibody or the multispecific anti-LRP6 antibody comprises HVR-H3 comprising the amino acid sequence NAVK (SEQ ID NO: 47). In one embodiment, the anti-LRP6 antibody or the multispecific anti-LRP6 antibody comprises HVR-H3 comprising the amino acid sequence NAIK (SEQ ID NO: 48). In one embodiment, the anti-LRP6 antibody or the multispecific anti-LRP6 antibody comprises HVR-H3 comprising the amino acid sequence NAVKN (SEQ ID NO: 49). In one embodiment, the anti-LRP6 antibody or the multispecific anti-LRP6 antibody comprises HVR-H3 comprising the amino acid sequence NAIKN (SEQ ID NO: 50).

In any of the above embodiments, the anti-LRP6 antibody is a humanized antibody. In one embodiment, the anti-LRP6 antibody comprises an HVR as in any of the above embodiments, and further comprises an acceptor human framework, such as a human immunoglobulin framework or human consensus framework.

In another aspect, the anti-LRP6 antibody is at least 90%, 91%, 92%, 93%, 94%, 95%, 96% (VH) sequence having a sequence identity of 100%, 97%, 98%, 99% or 100%. In yet another aspect, the anti-LRP6 antibody is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 90% identical to the amino acid sequence of SEQ ID NO: 9, SEQ ID NO: , 98%, 99% or 100% sequence identity to the amino acid sequence of the heavy chain variable domain (VH). In certain embodiments, a VH sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity is replaced For example, conservative substitutions), insertions or deletions, but the anti-LRP6 antibody comprising this sequence retains the ability to bind to LRP6. In a particular embodiment, a total of from 1 to 10 amino acids are substituted, inserted and / or deleted in the VH of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5 or SEQ ID NO: 7 or SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO:

In another aspect, the anti-LRP6 antibody is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% , 98%, 99% or 100% sequence identity. In certain embodiments, heavy chain sequences having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity are replaced For example, conservative substitutions), insertions or deletions, but the anti-LRP6 antibody comprising this sequence retains the ability to bind to LRP6. In certain embodiments, a total of 1 to 10 amino acids are substituted, inserted and / or deleted in SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5 or SEQ ID NO:

In certain embodiments, substitutions, insertions or deletions occur in the region outside the HVR (i.e., FR). Optionally, the anti-LRP6 antibody comprises a heavy chain and / or heavy chain VH of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5 or SEQ ID NO: 7, including post-translational modifications of the sequence.

In yet another aspect, at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% An anti-LRP6 antibody comprising a light chain variable domain (VL) having 98%, 99% or 100% sequence identity is provided. In yet another aspect, at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99 Lt; / RTI &gt; antibody comprising a light chain variable domain (VL) having a percent identity of 100% or 100%. In certain embodiments, a VL sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity, For example, conservative substitutions), insertions or deletions, but the anti-LRP6 antibody comprising this sequence retains the ability to bind to LRP6. In certain embodiments, a total of from 1 to 10 amino acids are substituted, inserted and / or deleted in a VL of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6 or SEQ ID NO: 8, or SEQ ID NO: 10, SEQ ID NO:

In another aspect, the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6 or SEQ ID NO: 8 is at least 90%, 91%, 92%, 93%, 94%, 95%, 96% Lt; / RTI &gt; antibody is provided comprising a light chain having a sequence identity of 100% or 100%. In certain embodiments, light chain sequences having an identity of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% For example, conservative substitutions), insertions or deletions, but the anti-LRP6 antibody comprising this sequence retains the ability to bind to LRP6. In certain embodiments, a total of 1 to 10 amino acids are substituted, inserted and / or deleted in SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6 or SEQ ID NO:

In certain embodiments, substitutions, insertions or deletions occur in the region outside the HVR (i.e., FR). Optionally, the anti-LRP6 antibody comprises a light chain and / or VL sequence of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6 or SEQ ID NO: 8, including post-translational modifications of the sequence.

In another aspect, there is provided an anti-LRP6 antibody comprising a VH as in any of the provided embodiments described above and a VL as in any of the provided embodiments.

In a further aspect, the invention provides an antibody that binds to the same epitope as the anti-LRP6 antibody provided herein. For example, in certain embodiments, the VH sequence of SEQ ID NO. 9 and the VL sequence of SEQ ID NO. 10, or the VH sequence of SEQ ID NO. 11 and the VL sequence of SEQ ID NO. 12, or the VH sequence of SEQ ID NO. 13 and the VL sequence of SEQ ID NO. An anti-LRP6 antibody selected from an anti-LRP6 antibody comprising the VH sequence of SEQ ID NO: 16 and the VL sequence of SEQ ID NO: 16. In one embodiment, the anti-LRP6 antibody binds to an epitope consisting of an amino acid residue in the E1-E2 region of LRP6. In one embodiment, the anti-LRP6 antibody binds to an epitope consisting of an amino acid residue in the E3-E4 region of LRP6. In one embodiment, the anti-LRP6 antibody is a bispecific antibody that binds to an epitope consisting of an amino acid residue present in the E1-E2 region of LRP6 and to an epitope consisting of an amino acid residue present in the E3-E4 region of LRP6 .

In one embodiment, the anti-LRP6 antibody binds to a conformational epitope comprising residues R28, E51, D52, V70, S71, E73, L95, S96, D98, E115, R141 and N185 of LRP6. In one embodiment, the anti-LRP6 antibody is selected from the group consisting of residues R28, E51, D52, V70, S71, E73, L95, S96, D98, E115, R141, N185, R29, W188, K202, P225, H226, S243 and F266 of LRP6 Lt; RTI ID = 0.0 &gt; epitope &lt; / RTI &gt;

In one embodiment, the anti-LRP6 antibody comprises at least one, at least two of the amino acid residues R28, E51, D52, V70, S71, E73, L95, S96, D98, E115, R141, and N185 of the E1? -Propeller of LRP6 At least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least eleven or both. In a further embodiment, the anti-LRP6 antibody further comprises at least one, at least two, at least three, at least four, at least five, at least six, or at least two of LRP6 residues R29, W188, K202, P225, H226, S243 and F266 Interact with at least seven dogs.

In a further aspect, the anti-LRP6 antibody according to any of the above embodiments may incorporate any feature singly or in combination, as described in the following sections 1-7:

1. Antibody affinity

In certain embodiments, the antibody provided herein is ≤ 1 μM, ≤ 100 nM, ≤ 10 nM, ≤ 1 nM, ≤ 0.1 nM, ≤ 0.01 nM , or ≤ 0.001 nM (e.g., 10 -8 M or less, e. (10 -8 M to 10 -13 M, for example, 10 -9 M to 10 -13 M) dissociation constants (Kd).

In one embodiment, Kd is measured by a radiolabeled antigen binding assay (RIA) performed using a Fab version of the antibody of interest and its antigen as described in the following assays. The solution binding affinity of the Fab for the antigen was determined by equilibrating the Fab with a minimal concentration of ( 125 I) -labeled antigen in the presence of a titration series of unlabeled antigens, and then conjugating the bound antigen to the anti-Fab antibody- (See, for example, Chen et al., J. Mol. Biol. 293: 865-881 (1999)). To establish assay conditions, a MICROTITER multi-well plate (Thermo Scientific) was incubated with 5 占 퐂 / ml of capture anti-Fab antibody (Capellaps (pH7.6) in 50 mM sodium carbonate Cappel Labs) overnight and then blocked with 2% (w / v) bovine serum albumin in PBS for 2 to 5 hours at room temperature (approximately 23 ° C). In a non-adsorptive plate (Nunc # 269620), 100 pM or 26 pM [ 125 I] -antigen is mixed with serial dilutions of the Fab of interest (see, for example, Presta et al., Cancer Res. 57 : 4593-4599 (1997)], an estimate of the anti-VEGF antibody, Fab-12). The Fab of interest is then incubated overnight, but can be incubated for a longer time (e.g., about 65 hours) to ensure equilibrium is reached. The mixture is then transferred to a capture plate and incubated at room temperature (for example, for 1 hour). Then, the solution was removed and washed 8 times the plate with 0.1% Polysorbate 20 (Tween ® -20) in PBS. 150 μl / well scintillant (MICROSCINT-20 ™; Packard) was added to the plate and the plate was incubated with a TOPCOUNT ™ gamma counter (Packard) for 10 minutes Lt; / RTI &gt; The concentration of each Fab providing less than 20% of the maximal binding is selected and used for competitive binding assays.

According to another embodiment, the Kd is biacore ( R ) -2000 or Biacore ( R) -3000 (Biacore &lt; ( R ) &gt;) at 25 ° C using an antigen CM5 chip immobilized, for example, (BIAcore, Inc., Piscataway, NJ) using a surface plasmon resonance assay. Briefly, a carboxymethylated dextran biosensor chip (CM5, Biacore, Inc.) Is mixed with N-ethyl-N '- (3- dimethylaminopropyl) -carbodiimide hydrochloride (EDC) Is activated with N-hydroxysuccinimide (NHS). The antigen is diluted to 5 μg / mL (about 0.2 μM) using 10 mM sodium acetate (pH 4.8) and then injected at a flow rate of 5 μl / min to achieve approximately 10 reaction units (RU) of the coupled protein. After injection of the antigen, 1M ethanolamine is injected to block the unreacted group. For kinetic measurements, two-fold serial dilutions of Fab (0.78 nM to 500 nM) were diluted in PBS containing 0.05% polysorbate 20 (Tween-20 ™) surfactant at 25 ° C. at a flow rate of about 25 μl / (PBST). A simple one-to-one Lang calculates Muir binding model using (Biacore ® Evaluation Software version 3.2) by fitting the association and dissociation sensorgram at the same time meeting rate (k on) and dissociation rates (k off). The equilibrium dissociation constant (Kd) is calculated as the ratio of k off / k on . For example, Chen et al., J. Mol. Biol. 293: 865-881 (1999). The on-rate by the surface-plasmon resonance test was 10 &lt; 6 &gt; M -1 s -1 , the on-rate can be measured using a spectrometer such as the 8000-series SLM-AMINCO (TM) with a static-flow setup spectrophotometer (Aviv Instruments) or with a stirring cuvette Antigenic antibody (Fab form) of 20 nM in PBS (pH 7.2) in the presence of increasing concentrations of antigen as measured on a spectrophotometer (ThermoSpectronic) has a fluorescence emission intensity at 25 캜 (excitation = 295 nm, emission = 340 nm, 16 nm passband).

2. Antibody fragments

In certain embodiments, the antibody provided herein is an antibody fragment. Antibody fragments include, but are not limited to, Fab, Fab ', Fab'-SH, F (ab') 2 , Fv and scFv fragments, and other fragments described below. For review of specific antibody fragments, see Hudson et al. Nat. Med. 9: 129-134 (2003). For review of scFv fragments, see, for example, Pluckthun, in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., (Springer-Verlag, New York), pp. 269-315 (1994); WO 93/16185; And U.S. Patent Nos. 5,571,894 and 5,587,458. For a discussion of Fab and F (ab &apos;) 2 fragments that contain salvage receptor binding epitope residues and have increased in vivo half life, see U.S. Pat. No. 5,869,046.

Diabodies are antibody fragments with two antigen-binding sites that can be bivalent or bispecific. See, for example, EP 404,097; WO 1993/01161; Hudson et al., Nat. Med. 9: 129-134 (2003); And Hollinger et al., Proc. Natl. Acad. Sci. USA 90: 6444-6448 (1993). Triabodies and tetrabodies are also described in Hudson et al., Nat. Med. 9: 129-134 (2003).

A single-domain antibody is an antibody fragment comprising all or part of the heavy chain variable domain of an antibody, or all or part of a light chain variable domain. In certain embodiments, the single-domain antibody is a human single-domain antibody (see Domantis, Inc., Waltham, Mass., E.g., U.S. Patent No. 6,248,516 B1).

Antibody fragments can be produced by a variety of techniques including, but not limited to, production by recombinant host cells (e. G. E. coli or phage) as well as proteolytic digestion of intact antibodies as described herein .

3. Chimeric and humanized antibodies

In certain embodiments, the antibody provided herein is a chimeric antibody. Certain chimeric antibodies are described, for example, in U.S. Patent Nos. 4,816,567; And Morrison et al., Proc. Natl. Acad. Sci. USA, 81: 6851-6855 (1984). In one example, a chimeric antibody comprises a non-human variable region (e. G., A variable region derived from a mouse, rat, hamster, rabbit or non-human primate, such as a monkey) and a human constant region. In a further example, a chimeric antibody is a " class switching " antibody in which the class or subclass is changed from that of the parent antibody. A chimeric antibody comprises its antigen-binding fragment.

In certain embodiments, the chimeric antibody is a humanized antibody. Typically, non-human antibodies are humanized to reduce immunogenicity to humans while maintaining the specificity and affinity of the parent non-human antibody. Generally, humanized antibodies comprise one or more variable domains in which the HVR, e.g., a CDR (or portion thereof) is derived from a non-human antibody and the FR (or portion thereof) is derived from a human antibody sequence. The humanized antibody may also optionally comprise at least a portion of a human constant region. In some embodiments, some FR residues of the humanized antibody may be replaced with corresponding residues from a non-human antibody (e. G., An antibody from which the HVR residue is derived), for example to restore or enhance antibody specificity or affinity .

Humanized antibodies and methods for their preparation are described, for example, in Almagro and Fransson, Front. Biosci. 13: 1619-1633 (2008)), for example in Riechmann et al., Nature 332: 323-329 (1988); Queen et al., Proc. Nat'l Acad. Sci. USA 86: 10029-10033 (1989); U.S. Patent Nos. 5,821,337, 7,527,791, 6,982,321, and 7,087,409; [Kashmiri et al., Methods 36: 25-34 (2005)] (SDR (a-CDR) grafting substrate); [Padlan, Mol. Immunol. 28: 489-498 (1991) (described " rescaling "); [Dall'Acqua et al., Methods 36: 43-60 (2005)] (referred to as "FR shuffling"); And Osbourn et al., Methods 36: 61-68 (2005) and Klimka et al., Br. J. Cancer, 83: 252-260 (2000) (describing a "guide selection" approach to FR shuffling).

The human framework region that can be used for humanization can be selected from framework regions selected using the " best-fit " method (see, for example, Sims et al. J. Immunol. 151: 2296 (1993)); A framework region (e. G., Carter et al. Proc. Natl. Acad. Sci. USA, 89: 4285 (1992)) derived from the consensus sequence of human subclasses of a particular subgroup of light or heavy chain variable regions Presta et al. J. Immunol., 151: 2623 (1993)); Human maturation (somatic cell maturation) framework regions or human wiring framework regions (see, for example, Almagro and Fransson, Front. Biosci. 13: 1619-1633 (2008)); (Baca et al., J. Biol. Chem. 272: 10678-10684 (1997) and Rosok et al., J. Biol. Chem. 271: 22611-22618 (1996)).

4. Human Antibody

In certain embodiments, the antibody provided herein is a human antibody. Human antibodies can be generated using a variety of techniques known in the art. Human antibodies are generally described in van Dijk and van de Winkel, Curr. Opin. Pharmacol. 5: 368-74 (2001) and Lonberg, Curr. Opin. Immunol. 20: 450-459 (2008).

Human antibodies can be produced by administering an immunogen to a transgenic animal that has been modified to produce an intact human antibody or an intact antibody with a human variable region in response to an antigen challenge. Such animals typically contain all or part of a human immunoglobulin locus that replaces the endogenous immunoglobulin locus, or that is extrachromosomally or randomly integrated into the chromosome of the animal. In these transgenic mice, the endogenous immunoglobulin locus is generally inactivated. For a review of methods for obtaining human antibodies from transgenic animals, see Lonberg, Nat. Biotech. 23: 1117-1125 (2005). See also, for example, U.S. Patent Nos. 6,075,181 and 6,150,584 (described by XENOMOUSE TM technology); U.S. Patent No. 5,770,429 (described in HuMab ® technology); See U.S. Patent No. 7,041,870 (KM mice (KM MOUSE) ® technology described), and U.S. Patent Application Publication No. US 2007/0061900 (Velocity mouse (VelociMouse) ® technology described). The human variable region from an intact antibody produced by such an animal may be further modified, for example, in combination with a different human constant region.

Human antibodies can also be prepared by hybridoma-based methods. Human myeloma and mouse-human xenogeneic myeloma cell lines for the production of human monoclonal antibodies are described. (Kozbor J. Immunol., 133: 3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc., New York, 1987); And Boerner et al., J. Immunol., 147: 86 (1991).) Human antibodies produced through human B-cell hybridoma techniques have also been described in Li et al., Proc. Natl. Acad. Sci. USA, 103: 3557-3562 (2006). Additional methods are described, for example, in U.S. Patent No. 7,189,826 (monoclonal human IgM antibody production from a hybridoma cell line) and in Ni, Xiandai Mianyixue, 26 (4): 265-268 (2006) - human hybridoma substrate). Human hybridoma technology (Trioma technology) is also described in Vollmers and Brandlein, Histology and Histopathology, 20 (3): 927-937 (2005) and Vollmers and Brandlein, Methods and Findings in Experimental and Clinical Pharmacology, 27 (3): 185-91 (2005).

Human antibodies can also be generated by isolating Fv clone variable domain sequences selected from a human-derived phage display library. Such variable domain sequences can then be combined with the preferred human constant domains. Techniques for selecting human antibodies from antibody libraries are described below.

5. Library-derived antibodies

The antibodies of the present invention can be isolated by screening combinatorial libraries for antibodies having the desired activity or activities. Various methods are known in the art for generating phage display libraries, for example, and screening such libraries for antibodies with desirable binding properties. Such a method is described, for example, in Hoogenboom et al. for example, in McCafferty et al., Nature 348: 552-7 (1986)), which has been reviewed in &lt; RTI ID = 0.0 &554; Clackson et al., Nature 352: 624-628 (1991); Marks et al., J. Mol. Biol. 222: 581-597 (1992); Marks and Bradbury, in Methods in Molecular Biology 248: 161-175 (Lo, ed., Human Press, Totowa, NJ, 2003); Sidhu et al., J. Mol. Biol. 338 (2): 299-310 (2004); Lee et al., J. Mol. Biol. 340 (5): 1073-1093 (2004); Fellouse, Proc. Natl. Acad. Sci. USA 101 (34): 12467-12472 (2004); And Lee et al., J. Immunol. Methods 284 (1-2): 119-132 (2004).

In a particular phage display method, the repertoires of VH and VL genes are individually cloned by polymerase chain reaction (PCR) and randomly recombined into a phage library, which is described in Winter et al., Ann. Rev. Immunol., 12: 433-455 (1994). Phage typically displays antibody fragments as single-chain Fv (scFv) fragments or Fab fragments. Libraries from immunized sources provide high-affinity antibodies to immunogens without the need to build hybridomas. Alternatively, a naïve repertoire can be cloned (e.g., from a human) to generate a broad range of non-autologous and / or transcriptional modifications without any immunization as described in Griffiths et al., EMBO J, 12: 725-734 It can also provide a single source of antibodies to autoantigens. Finally, as described in Hoogenboom and Winter, J. Mol. (SEQ ID NO: 2), cloning V rearranged V-gene fragments from stem cells, coding for highly variable CDR3 regions and in vitro sequencing to achieve in vitro rearrangement, as described in Biol., 227: 381-388 Lt; RTI ID = 0.0 &gt; a &lt; / RTI &gt; PCR primer. Patent publications describing human antibody phage libraries are described, for example, in U.S. Patent Nos. 5,750,373 and U.S. Patent Publication Nos. 2005/0079574, 2005/0119455, 2005/0266000, 2007/0117126, 2007/0160598, 2007/0237764, 2007 / 0292936 and 2009/0002360.

Antibodies or antibody fragments isolated from human antibody libraries are herein considered human antibodies or human antibody fragments.

6. Multispecific antibodies

In certain embodiments, the antibodies provided herein are multispecific antibodies, e. G. Bispecific antibodies. A multispecific antibody is a monoclonal antibody having binding specificities for at least two different sites. In certain embodiments, one of the binding specificities is for LRP6 and the other is for any other antigen. In certain embodiments, bispecific antibodies bind to two different epitopes of LRP6. Bispecific antibodies can also be used to locate cytotoxic agents in cells expressing LRP6. Bispecific antibodies can be produced as whole antibody or antibody fragments.

Techniques for producing multispecific antibodies include recombinant co-expression of two immunoglobulin heavy chain-light chain pairs with different specificity (Milstein and Cuello, Nature 305: 537 (1983)), WO 93/08829, And "knob-in-hole" manipulations (see, for example, U.S. Patent No. 5,731,168), and Traunecker et al., EMBO J. 10: 3655 (1991) Multispecific antibodies also include manipulating electrostatic steering effects to produce antibody Fc-heterodimeric molecules (WO 2009 / 089004A1); Cross-linking of two or more antibodies or fragments (see, for example, U.S. Patent No. 4,676,980 and Brennan et al., Science, 229: 81 (1985)); The use of leucine zipper to produce bispecific antibodies (see, for example, Kostelny et al., J. Immunol., 148 (5): 1547-1553 (1992)); (See, for example, Hollinger et al., Proc. Natl. Acad. Sci. USA, 90: 6444-6448 (1993)) for the preparation of bispecific antibody fragments; And the use of single-chain Fv (sFv) dimers (see, for example, Gruber et al., J. Immunol., 152: 5368 (1994)); And for example, Tutt et al. J. Immunol. 147: 60 (1991)). &Lt; RTI ID = 0.0 &gt;

Engineered antibodies with three or more functional antigen binding sites, including " octopus antibodies ", are also included herein (see, e.g., US 2006/002576A1).

The antibody or fragment herein also includes a " dual-acting FAb " or " DAF " comprising an antigen binding site that binds LRP6 as well as another, different antigen (see, e.g., US 2008/0069820).

7. Antibody variants

In certain embodiments, amino acid sequence variants of the antibodies provided herein are contemplated. For example, it may be desirable to improve the binding affinity and / or other biological properties of the antibody. Amino acid sequence variants of the antibody may be prepared by introducing appropriate modifications to the nucleotide sequence encoding the antibody or by peptide synthesis. Such modifications include, for example, deletion and / or insertion and / or substitution of residues within the amino acid sequence of the antibody. Any combination of deletion, insertion and substitution can be made to arrive at the final construct so that the final construct retains the desired properties, e. G. Antigen-binding.

a) substitution, insertion and deletion mutants

In certain embodiments, antibody variants having one or more amino acid substitutions are provided. Interest regions for inducing replacement mutations include HVR and FR. Conservative substitutions are shown in Table 1 under the heading " Conservative substitutions ". More substantial changes are provided as further described below with respect to amino acid side-chain classes in Table 1 under the heading " Exemplary Substitutions ". Amino acid substitutions are introduced into the antibody of interest and the product can be screened for the desired activity, for example, maintenance / improved antigen binding, reduced immunogenicity or improved ADCC or CDC.

Figure 112012086169669-pct00001

Figure 112012086169669-pct00002

Amino acids can be classified according to their common side chain properties:

(1) hydrophobicity: norleucine, Met, Ala, Val, Leu, Ile;

(2) Neutral hydrophilic: Cys, Ser, Thr, Asn, Gln;

(3) Acid: Asp, Glu;

(4) Basicity: His, Lys, Arg;

(5) residues affecting the chain orientation: Gly, Pro;

(6) Aromatic: Trp, Tyr, Phe.

Non-conservative substitutions will involve exchanging members of one of these classes for another class.

One type of substitutional variant involves substituting one or more hypervariable region residues of a parent antibody (e. G., A humanized or human antibody). Generally, the generated variant (s) selected for further study will have a modification (e. G., Improvement) of specific biological properties (e. G., Increased affinity, reduced immunogenicity) Or substantially retain certain biological properties of the parent antibody. Exemplary substitution variants are affinity maturation antibodies that can be conveniently generated using, for example, phage display-based affinity maturation techniques such as those described herein. Briefly, one or more HVR residues are mutated, variant antibodies are displayed on a phage, and screened for a particular biological activity (e. G., Binding affinity).

Alterations (e.g., substitutions) can be made in the HVR, for example, to improve antibody affinity. These changes may result in an HVR " hotspot, " a binding affinity during the somatic cell maturation process (see, e.g., Chowdhury, Methods Mol. Biol. 207: 179-196 (2008) In a residue encoded by a codon that performs mutation at a high frequency with the resulting variant VH or VL tested against B. Affinity maturation by construction and reselection from a secondary library is described, (O'Brien et al., Ed., Human Press, Totowa, NJ, (2001)]. In some embodiments of affinity maturation, , Diversity is introduced into a variable gene selected for maturation by any of a variety of methods (e. G., Error-triggered PCR, chain shuffling, or oligonucleotide-induced mutagenesis). Secondary antibodies are then generated . Then, any antibody with the desired affinity Other methods of introducing diversity are associated with the HVR-directed approach, where multiple HVR residues (e.g., 4 to 6 residues at a time) are randomized. HVR residues associated with binding can be specifically identified using, for example, alanine scanning mutagenesis or modeling. In particular, CDR-H3 and CDR-L3 are often targeted.

In certain embodiments, substitution, insertion or deletion can occur within one or more HVRs, so long as such alteration does not substantially reduce the ability of the antibody to bind to the antigen. For example, conservative modifications (e. G., Conservative substitutions as provided herein) that do not substantially reduce binding affinity can be made in the HVR. These changes may be outside the HVR " hotspot " or the SDR. In certain embodiments of the variant VH and VL sequences provided above, each HVR is unaltered, or contains no more than 1, 2, or 3 amino acid substitutions.

A useful method for identifying a residue or region of an antibody that can be targeted for mutagenesis, as described in Cunningham and Wells (1989) Science, 244: 1081-1085, is referred to as " alanine scanning mutagenesis. &Quot; In this method, a group of residues or target residues is identified (e.g., charged residues such as arg, asp, his, lys and glu), neutral or negatively charged amino acids (e.g., alanine or polyalanine ) To determine whether it affects the interaction of the antibody with the antigen. Additional substitutions may be introduced at amino acid positions that demonstrate functional susceptibility to initial substitution. Alternatively or additionally, it may be beneficial to analyze the crystal structure of the antigen-antibody complex to determine the contact point between the antibody and the antigen. Such contact residues and neighboring residues can be targeted or removed as candidates for substitution. Variants can be screened to determine whether they contain desirable properties.

Amino acid sequence insertions include amino- and / or carboxyl-terminal fusions ranging in length ranging from one residue to more than 100 residues in the polypeptide, as well as sequential insertion of single or multiple amino acid residues. Examples of terminal insertions include antibodies having an N-terminal methionyl residue. Other insertional variants of the antibody molecule include those in which an enzyme (e.g., in the case of ADEPT) or a polypeptide that increases the serum half-life of the antibody is fused to the N- or C-terminus of the antibody.

b) glycosylation variants

In certain embodiments, the antibodies provided herein are modified to increase or decrease the extent to which the antibody is glycosylated. Addition or deletion of the glycosylation site to the antibody can conveniently be accomplished by altering the amino acid sequence so that one or more glycosylation sites are created or removed.

Where the antibody comprises an Fc region, the carbohydrate attached thereto may be altered. Natural antibodies produced by mammalian cells typically include branched, branched oligosaccharides that are typically attached by N-linkage to Asn297 of the CH2 domain of the Fc region. See, for example, Wright et al. TIBTECH 15: 26-32 (1997). Oligosaccharides may include various carbohydrates such as mannose, N-acetylglucosamine (GlcNAc), galactose and sialic acid as well as fucose attached to GlcNAc in the " stem " of this branched oligosaccharide structure. In some embodiments, modification of the oligosaccharides in the antibodies of the invention can be made to produce antibody variants with certain improved properties.

In one embodiment, antibody variants having carbohydrate structures lacking fucose attached (directly or indirectly) to the Fc region are provided. For example, the amount of fucose in such antibodies may be between 1% and 80%, between 1% and 65%, between 5% and 65%, or between 20% and 40%. The amount of fucose is compared to the sum of all sugar structures (e.g., complexes, hybrids and gonorrhea structures) attached to Asn 297 as measured by MALDI-TOF mass spectrometry as described, for example, in WO 2008/077546 Is determined by calculating the average amount of fucose in the sugar chains in Asn297. Asn297 represents an asparagine residue located at the weak position 297 of the Fc region (Eu numbering of Fc region residues); Asn297 can also be located upstream or downstream of about +/- 3 amino acids of position 297, i.e., between positions 294 and 300, by additional sequence modification of the antibody. Such fucosylation variants may have improved ADCC function. For example, U.S. Patent Publication No. US 2003/0157108 (Presta, L.); US 2004/0093621 (Kyowa Hakko Kogyo Co., Ltd.). Examples of references to " tamifucosyl " or " fucose-deficient " antibody variants include: US 2003/0157108; WO 2000/61739; WO 2001/29246; US 2003/0115614; US 2002/0164328; US 2004/0093621; US 2004/0132140; US 2004/0110704; US 2004/0110282; US 2004/0109865; WO 2003/085119; WO 2003/084570; WO 2005/035586; WO 2005/035778; WO2005 / 053742; WO2002 / 031140; Okazaki et al. J. Mol. Biol. 336: 1239-1249 (2004); Yamane-Ohnuki et al. Biotech. Bioeng. 87: 614 (2004)). Examples of cell lines capable of producing the dedufucosylated antibody include Lecl3 CHO cells lacking protein fucosylation (Ripka et al. Arch. Biochem. Biophys. 249: 533-545 (1986)); U.S. Patent Application No. US 2003 Such as alpha-1,6-fucosyltransferase gene, FUT8, knockout CHO cells (e. G. (For example, Yamane-Ohnuki et al., Biotech. Bioeng. 87: 614 (2004); Kanda, Y. et al., Biotechnol. Bioeng., 94 (4): 680-688 (2006); And WO 2003/085107).

Additional antibody variants with bisecting oligosaccharides are provided, for example, the branched oligosaccharides attached to the Fc region of the antibody are bisected by GlcNAc. Such antibody variants can reduce fucosylation and / or improve ADCC function. Examples of such antibody variants are described, for example, in WO 2003/011878 (Jean-Mairet et al.); U.S. Patent No. 6,602,684 (Umana et al.); And US 2005/0123546 (Umana et al.). Antibody variants having at least one galactose residue in the oligosaccharide attached to the Fc region are also provided. Such antibody variants may have improved CDC function. Such antibody variants are described, for example, in WO 1997/30087 (Patel et al.); WO 1998/58964 (Raju, S.); And WO 1999/22764 (Raju, S.).

c) Fc region variants

In certain embodiments, one or more amino acid modifications may be introduced into the Fc region of the antibody provided herein to generate Fc region variants. Fc region variants may comprise a human Fc region sequence (e.g., a human IgG1, IgG2, IgG3, or IgG4 Fc region) comprising an amino acid modification (e.g., substitution) at one or more amino acid positions.

In certain embodiments, the present invention is not all effector functions, but it has several effector functions, so that although the half-life of the antibody in vivo is important, certain effector functions (such as complement and ADCC) &Lt; / RTI &gt; antibody variants that are preferred candidates for the antibody. In vitro and / or in vivo cytotoxicity assays can be performed to confirm reduction / depletion of CDC and / or ADCC activity. For example, an Fc receptor binding (FcR) binding assay can be performed to confirm that the antibody does not have an Fc [gamma] R binding (thus, presumably lacking ADCC activity) and retains FcRn binding capacity. NK cells, the primary cells mediating ADCC, express only Fc [gamma] RIIII whereas monocytes express Fc [gamma] RI, Fc [gamma] RII and Fc [gamma] RIII. FcR expression on hematopoietic cells is described in Ravetch and Kinet, Annu. Rev. Immunol. 9: 457-492 (1991), page 464, Table 3. A non-limiting example of an in vitro assay for assessing ADCC activity of a molecule of interest is described in U.S. Patent No. 5,500,362 (e.g., Hellstrom, I. et al. Proc. Nat'l Acad. Sci. USA 83: 7059 -7063 (1986) and Hellstrom, I et al., Proc. Nat'l Acad. Sci. USA 82: 1499-1502 (1985)); 5,821,337 (Bruggemann, M. et al., J. Exp. Med. 166: 1351-1361 (1987)). Alternatively, non-radioactive assay methods can be used (see, for example, ACTI) non-radioactive cytotoxicity assays for flow cytometry (CellTechnology, Inc., Mountain, CA ; And CytoTox 96 ® non-radioactive cytotoxicity assays (Promega, Madison, Wis.)). Effector cells useful for such assays include peripheral blood mononuclear cells (PBMC) and natural killer (NK) cells. Alternatively or additionally, the ADCC activity of the molecule of interest can be determined in vivo, for example, in Clynes et al. Proc. Nat'l Acad. Sci. USA 95: 652-656 (1998). In addition, a C1q binding assay can be performed to confirm that the antibody is unable to bind to C1q and thus lacks CDC activity. See, for example, the C1q and C3c binding ELISAs of WO 2006/029879 and WO 2005/100402. CDC assays can be performed to assess complement activation (see, for example, Gazzano-Santoro et al., J. Immunol. Methods 202: 163 (1996); Cragg, MS et al., Blood 101: 1045 -1052 (2003); and Cragg, MS and MJ Glennie, Blood 103: 2738-2743 (2004)). FcRn binding and in vivo removal rate / half-life determination can also be performed using methods known in the art (see, for example, Petkova, SB et al., Int'l. Immunol. 18 (12): 1759 -1769 (2006)).

Antibodies with reduced effector function include those having one or more substitutions of Fc region residues 238, 265, 269, 270, 297, 327 and 329 (US Patent No. 6,737,056). Such an Fc mutant has an Fc mutant having a substitution at two or more of the amino acid positions 265, 269, 270, 297 and 327 (including the so-called " DANA " Fc mutant with substitution with alanine at residues 265 and 297) (U.S. Patent No. 7,332,581).

Certain antibody variants having improved or decreased binding to FcR are described. (See, for example, U.S. Patent No. 6,737,056; WO 2004/056312; and Shields et al., J. Biol. Chem. 9 (2): 6591-6604 (2001)).

In certain embodiments, antibody variants comprise one or more amino acid substitutions that improve ADCC, such as Fc regions with substitutions at positions 298, 333 and / or 334 (EU numbering of residues) of the Fc region.

In some embodiments, for example, U.S. Patent No. 6,194,551, WO 99/51642, and Idusogie et al. J. Immunol. 164, 4178-4184 (2000)), changes are made in the Fc region that represent altered (i.e., improved or reduced) C1q binding and / or complement dependent cytotoxicity (CDC).

(FcRn) (Guyer et al., J. Immunol. 117: 587 (1976) and Kim et al., J. Immunol. 24: 249 (1994)), which is responsible for transferring maternal IgG to the fetus. ]) And improved half-life are described in US 2005/141993 A1 (Hinton et al.). These antibodies comprise an Fc region having one or more substitutions that improve the binding of the Fc region to the FcRn. Such an Fc variant may be selected from the group consisting of Fc region residues: 238, 256, 265, 272, 286, 303, 305, 307, 311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, 413, 434, e.g. having a substitution of the Fc region residue 434 (U. S. Patent No. 7,371, 826).

Other examples of Fc region variants are also described in Duncan & Winter, Nature 322: 738-40 (1988); U.S. Patent No. 5,648,260; U.S. Patent No. 5,624,821; And WO 94/29351.

d) Cysteine engineered antibody variants

In certain embodiments, it may be desirable to prepare a cysteine engineered antibody, e.g., a "thio MAb," in which one or more residues of the antibody are replaced with a cysteine residue. In certain embodiments, such substituted moieties occur at accessible sites of the antibody. Substituting these residues for cysteine places the reactive thiol group at the accessible site of the antibody, which can be used to conjugate the antibody to another moiety, such as a drug moiety or linker-drug moiety, as further described herein A conjugate can be generated. In certain embodiments, any one or more of the following residues may be substituted with cysteine: V205 of light chain (Kabat numbering); A118 of the heavy chain (EU numbering); And S400 (EU numbering) of the heavy chain Fc region. Cysteine engineered antibodies can be produced, for example, as described in U.S. Patent No. 7,521,541.

e) Antibody derivatives

In certain embodiments, the antibodies provided herein can be further modified to contain additional non-proteinaceous moieties that are well known in the art and readily available. Suitable moieties for derivatization of antibodies include, but are not limited to, water soluble polymers. Non-limiting examples of water soluble polymers include polyethylene glycol (PEG), copolymers of ethylene glycol / propylene glycol, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, poly- Ethylene / maleic anhydride copolymers, polyamino acids (homopolymers or random copolymers), and dextran or poly (n-vinylpyrrolidone) polyethylene glycols, propylene glycol homopolymers, But are not limited to, polypropylene oxide / ethylene oxide copolymers, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof. Polyethylene glycol propionaldehyde may have advantages in manufacturing due to its stability in water. The polymer may have any molecular weight and may be branched or unbranched. The number of polymers attached to the antibody can vary, and when more than one polymer is attached, the polymers can be the same or different molecules. In general, the number and / or types of polymers used in derivatization will depend on a number of considerations including, but not limited to, the particular characteristics or functions of the antibody to be ameliorated, whether or not the antibody derivative will be used in therapy under defined conditions, . &Lt; / RTI &gt;

In another embodiment, a conjugate of an antibody and a non-proteinaceous moiety that can be selectively heated by radiation exposure is provided. In one embodiment, the non-proteinaceous moiety is a carbon nanotube (Kam et al., Proc. Natl. Acad. Sci. USA 102: 11600-11605 (2005)). The radiation may be of any wavelength, including, but not limited to, wavelengths that heat non-proteinaceous moieties to a temperature that does not harm normal cells but which is close to the antibody-non-proteinaceous moiety.

B. Recombinant Methods and Compositions

Antibodies can be produced using recombinant methods and compositions, for example, as described in U.S. Patent No. 4,816,567. In one embodiment, isolated nucleic acids encoding the anti-LRP6 antibodies described herein are provided. Such a nucleic acid may encode an amino acid sequence comprising the VL of the antibody and / or an amino acid sequence comprising the VH (e.g., the light and / or heavy chain of the antibody). In a further embodiment, one or more vectors (e. G., Expression vectors) comprising such nucleic acids are provided. In a further embodiment, host cells comprising such nucleic acids are provided. In one such embodiment, the host cell comprises (1) a vector comprising a nucleic acid encoding an amino acid sequence comprising the amino acid sequence comprising the VL of the antibody and the VH of the antibody, or (2) a vector comprising the amino acid sequence comprising the VL of the antibody (E. G., Transfected with) a first vector comprising a nucleic acid encoding a VH of the antibody and a nucleic acid encoding an amino acid sequence comprising the VH of the antibody. In one embodiment, the host cell is eukaryotic, for example Chinese hamster ovary (CHO) cells or lymphoid cells (e.g., Y0, NS0, Sp20 cells). In one embodiment, a host cell comprising a nucleic acid encoding an anti-LRP6 antibody as provided above is cultivated under conditions suitable for expression of the antibody, and the antibody is optionally recovered from the host cell (or host cell culture medium) Lt; RTI ID = 0.0 &gt; anti-LRP6 &lt; / RTI &gt;

For recombinant production of an anti-LRP6 antibody, for example, the nucleic acid encoding the antibody as described above is isolated and inserted into one or more vectors for further cloning and / or expression in host cells. Such nucleic acids can be readily isolated and sequenced using conventional procedures (e. G., By using oligonucleotide probes that are capable of specifically binding to the genes encoding the heavy and light chains of the antibody).

Suitable host cells for cloning or expression of the antibody-coding vector include the prokaryotic or eukaryotic cells described herein. For example, antibodies may be produced in bacteria, especially when glycosylation and Fc effector function is not required. For the expression of antibody fragments and polypeptides in bacteria, see, for example, U.S. Patent Nos. 5,648,237, 5,789,199 and 5,840,523. (See also Charlton, Methods in Molecular Biology, Vol. 248 (BKC Lo, ed., Humana Press, Totowa, NJ, 2003), pp. 245-254, which describes the expression of antibody fragments in E. coli ). After expression, the antibody can be isolated from the bacterial cell paste in a soluble fraction and further purified.

In addition to prokaryotes, eukaryotic microbes such as filamentous fungi or yeasts, including fungal and yeast strains, which cause the glycosylation pathway to " humanize " to produce antibodies in partial or total human glycosylation patterns, It is suitable for expression. Gerngross, Nat. Biotech. 22: 1409-1414 (2004), and Li et al., Nat. Biotech. 24: 210-215 (2006).

Suitable host cells for expression of glycosylated antibodies are also derived from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells include plant and insect cells. A number of baculovirus strains have been identified that can be used to transfect insect cells, particularly Spodoptera frugiperda cells.

Plant cell cultures can also be used as hosts. See, for example, U.S. Patent Nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978 and 6,417,429 (describing PLANTIBODIES TM technology for producing antibodies in transgenic plants).

Vertebrate cells can also be used as hosts. For example, mammalian cell lines suitable for growing in suspension may be useful. Other examples of useful mammalian host cell lines are the monkey kidney CV1 cell line (COS-7) transformed by SV40; Human embryonic kidney cell lines (e.g., 293 or 293 cells as described in Graham et al., J. Gen Virol. 36:59 (1977)); Fetal hamster kidney cells (BHK); Mouse Sertoli cells (for example, TM4 cells described in Mather, Biol. Reprod. 23: 243-251 (1980)); Monkey kidney cells (CV1); African green monkey kidney cells (VERO-76); Human cervical carcinoma cells (HELA); Canine kidney cells (MDCK); Buffalo rat liver cells (BRL 3A); Human lung cells (W138); Human liver cells (Hep G2); Mouse breast tumor (MMT 060562); See, e.g., Mather et al., Annals NY Acad. Sci. 383: 44-68 (1982); MRC 5 cells; And FS4 cells. Other useful mammalian host cell lines include Chinese hamster ovary (CHO) cells, such as DHFR - CHO cells (Urlaub et al., Proc. Natl. Acad. Sci. USA 77: 4216 (1980)); And myeloma cell lines such as Y0, NS0 and Sp2 / 0. For review of specific mammalian host cell lines suitable for antibody production, see, for example, Yazaki and Wu, Methods in Molecular Biology, Vol. 248 (BKC Lo, ed., Humana Press, Totowa, N.J.), pp. 255-268 (2003).

C. Black

The anti-LRP6 antibodies provided herein can be identified by various assays known in the art, screened, or characterized for their physical / chemical properties and / or biological activity.

1. Combination test and other test

In one aspect, the antibodies of the invention are tested for their antigen binding activity by known methods, such as, for example, ELISA, Western blot, and the like.

In another aspect, competition assays can be used to identify antibodies that compete with the anti-LRP6 antibodies of the invention for binding to LRP6. In certain embodiments, such competing antibodies bind to the same epitope (e. G., A linear or conformational epitope) bound by the anti-LRP6 antibody of the invention. Details of exemplary methods for mapping epitopes to which antibodies bind are described in Morris (1996) " Epitope Mapping Protocols, " in Methods in Molecular Biology, vol. 66 (Humana Press, Totowa, NJ).

In an exemplary competition assay, a first labeled antibody that binds fixed LRP6 to LRP6 and a second unlabeled antibody that is tested for its ability to compete with the first antibody for binding to LRP6 is incubated . The second antibody may be present in the hybridoma supernatant. As a control, immobilized LRP6 is incubated in a solution containing the first labeled antibody and not containing the second unlabeled antibody. After incubation under conditions permitting binding of the first antibody to LRP6, an excess amount of unbound antibody is removed and the amount of label associated with immobilized LRP6 is determined. If the amount of label associated with immobilized LRP6 is substantially reduced in the test sample relative to the control sample, this indicates that the second antibody competes with the first antibody for binding to LRP6. See Harlow and Lane (1988) Antibodies: A Laboratory Manual ch.14 (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY).

2. Active black

In one aspect, assays are provided to identify its anti-LRP6 antibodies with biological activity. Biological activities may include, for example, inhibition or enhancement of Wnt isoform mediated signaling, controlled bone mass / content, inhibition of cell proliferation, and increased cell proliferation. Antibodies with these biological activities are also provided in vivo and / or in vitro.

In certain embodiments, the antibodies of the invention are tested for such biological activity. Specific assays used to test biological activity are provided in the Examples.

D. Immunoconjugates

The present invention also relates to a method of treating or preventing a disease or disorder associated with one or more cytotoxic agents such as chemotherapeutic agents or drugs, growth inhibitors, toxins (e.g., an enzyme active toxin of bacterial, fungal, plant or animal origin or a fragment thereof) Lt; RTI ID = 0.0 &gt; anti-LRP6 &lt; / RTI &gt;

 In one embodiment, the immunoconjugate comprises an antibody selected from the group consisting of maytansinoids (see U.S. Patent Nos. 5,208,020, 5,416,064 and EP 0 425 235 B1); Auristatin such as monomethylauristatin drug moiety DE and DF (MMAE and MMAF) (see U.S. Patent Nos. 5,635,483 and 5,780,588 and 7,498,298); Dolastatin; Cancer Res. 53: 3336-3342 (1993); and Lode et al., U. S. Patent No. 5,714, , Cancer Res. 58: 2925-2928 (1998)); Anthracyclines such as daunomycin or doxorubicin (Kratz et al., Current Med. Chem. 13: 477-523 (2006); Jeffrey et al., Bioorganic & Med. Chem. Letters 16: 358-362 Proc Natl Acad Sci USA 97: 829-834 (2000); Dubowchik et al., Bioorg &lt; (R) &gt; King et al., J. Med. Chem. 45: 4336-4343 (2002); and U.S. Patent No. 6,630,579); Methotrexate; Bindeseo; Taxanes such as docetaxel, paclitaxel, laurotaxel, teflon cells and hortata taxol; Tricothecene; And an antibody-drug conjugate (ADC) conjugated to one or more drugs including but not limited to CC1065.

In another embodiment, the immunoconjugate is an enzyme active toxin or a fragment thereof (diphtheria A chain, unbound active fragment of diphtheria toxin, exotoxin A chain (derived from Pseudomonas aeruginosa), lysine A chain, Aleurites fordii protein, Dianthin protein, Phytolaca americana protein (PAPI, PAPII and PAP-S), Momordin A protein, Aleurites fordii protein, But are not limited to, monocarcinoma inhibitors, momordica charantia inhibitors, curcin, crotin, sapaonaria officinalis inhibitors, gelonins, mitogelins, resorcinosine, penomesins, enomycins and tricothecenes Including, but not limited to, the antibody described herein.

In another embodiment, the immunoconjugate comprises an antibody as described herein conjugated to a radioactive atom to form a radioactive conjugate. A variety of radioactive isotopes are available for the production of radioactive conjugates. Examples include At 211 , I 131 , I 125 , Y 90 , Re 186 , Re 188 , Sm 153 , Bi 212 , P 32 , Pb 212 , and radioactive isotopes of Lu. The radioactive conjugate may comprise radioactive atoms for scintillography studies, for example tc99m or I123 when used for detection, or a spin label for nuclear magnetic resonance (NMR) imaging (also known as magnetic resonance imaging, mri) Again iodine-123, iodine-131, indium-111, fluorine-19, carbon-13, nitrogen-15, oxygen-17, gadolinium, manganese or iron.

Antibodies and cytotoxic agents may be conjugated to various bifunctional protein coupling agents such as N-succinimidyl-3- (2-pyridyldithio) propionate (SPDP), succinimidyl- Methyl dicyclohexyl-1-carboxylate (SMCC), iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl adipimidate HCl), active esters Diazonium derivatives such as bis- (p-diazonium benzoyl) -ethylenediamine), aldehydes (e.g., glutaraldehyde), bis-azido compounds (e.g., bis Diisocyanates such as toluene 2,6-diisocyanate, and bis-activated fluorine compounds such as 1,5-difluoro-2,4-dinitrobenzene. For example, ricin immunotoxins can be prepared as described in Vitetta et al., Science 238: 1098 (1987). Carbon-14-labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriamine pentaacetic acid (MX-DTPA) is an exemplary chelating agent for conjugating a radioactive nucleotide to an antibody. See WO 94/11026. The linker may be a " cleavable linker " that facilitates release of the cytotoxic drug in the cell. For example, an acid labile linker, a peptidase-sensitive linker, a photodegradable linker, a dimethyl linker or a disulfide-containing linker (Chari et al., Cancer Res. 52: 127-131 No. 5,208, 020) can be used.

The immunoconjugates or ADCs of this disclosure may be used in combination with commercially available crosslinking-linker reagents (e.g., Pierce Biotechnology, Inc .; SMPB, SMPH, Sulfo-EMCS, Sulfo-GMBS, Sulfo-KMUS, Sulfo-MBS, Sulfo-SIB, Sulfo-SMCC, and Sulfo-SMPB, And SVSB (succinimidyl- (4-vinylsulfone) benzoate)), but are not limited to these conjugates.

E. Methods and compositions for diagnosis and detection

In certain embodiments, any anti-LRP6 antibody provided herein is useful for detecting the presence of LRP6 in a biological sample. The term " detection " as used herein includes quantitative or qualitative detection. In certain embodiments, the biological sample comprises cells or tissues.

In one embodiment, an anti-LRP6 antibody is provided for use in a diagnostic or detection method. In a further aspect, a method is provided for detecting the presence of LRP6 in a biological sample. In certain embodiments, the method comprises contacting a biological sample with an anti-LRP6 antibody as described herein under conditions that allow binding of the anti-LRP6 antibody to LRP6, and determining whether a complex is formed between the anti-LRP6 antibody and LRP6 / RTI &gt; Such methods may be in vitro or in vivo methods. In one embodiment, the anti-LRP6 antibody is used to select a subject that is eligible for therapy using an anti-LRP6 antibody, for example when LRP6 is a biomarker for patient selection.

Exemplary disorders that may be diagnosed using the antibodies of the invention include disorders of the cancer and skeletal system.

In certain embodiments, a labeled anti-LRP6 antibody is provided. The label may be a moiety detected directly or indirectly through, for example, an enzymatic or molecular interaction, such as a label or moiety (e.g., fluorescence, color, electron-dense, chemiluminescent and radioactive label) But are not limited to, enzymes or ligands. Exemplary labels include radioisotopes 32 P, 14 C, 125 I, 3 H and 131 I, fluorescent moieties such as rare earth chelates or fluorescein and derivatives thereof, rhodamine and derivatives thereof, dansyl, umbelliferone, Beta] -glucosidase inhibitors, such as ferulic acid, such as firefly luciferase and bacterial luciferase (US Patent No. 4,737,456), luciferin, 2,3-dihydropthalazine dione, horseradish peroxidase (HRP), alkaline phosphatase, Enzymes that use hydrogen peroxide to oxidize a dye precursor, such as, for example, glucose oxidase, galactosoxidase, and glucose-6-phosphate dehydrogenase, such as, for example, glucose oxidase, galactosidase, glucoamylase, lysozyme, saccharide oxidase, HRP, lactoperoxidase, or heterocyclic oxidases coupled with microperoxidases, such as, for example, nuclease and xanthine oxidase, biotin / avidin, spin labels, Includes a gripping cover, stable free radical such as but not limited to one.

F. Pharmaceutical formulations

Pharmaceutical formulations of an anti-LRP6 antibody as described herein may be prepared by incorporating such antibodies having the desired degree of purity into one or more pharmaceutically acceptable carriers (Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980) ) In the form of a lyophilized preparation or an aqueous solution. Pharmaceutically acceptable carriers are nontoxic to the recipient at the dosages and concentrations generally employed and include buffers such as phosphate, citrate, and other organic acids; Antioxidants such as ascorbic acid and methionine; A preservative such as octadecyldimethylbenzylammonium chloride, hexamethonium chloride, benzalkonium chloride, benzethonium chloride, phenol, butyl or benzyl alcohol, alkyl parabens such as methyl or propyl paraben, catechol, resorcinol, cyclohexane 3-pentanol; and m-cresol); Low molecular weight (less than about 10 residues) polypeptides; Proteins such as serum albumin, gelatin or immunoglobulin; Hydrophilic polymers such as polyvinylpyrrolidone; Amino acids such as glycine, glutamine, asparagine, histidine, arginine or lysine; Monosaccharides, disaccharides and other carbohydrates such as glucose, mannose or dextrin; Chelating agents such as EDTA; Sugars such as sucrose, mannitol, trehalose or sorbitol; Salt-forming counter-ions such as sodium; Metal complexes (e. G., Zn-protein complexes); And / or non-ionic surfactants such as polyethylene glycol (PEG). Exemplary pharmaceutically acceptable carriers herein are also interstitial drug dispersants such as soluble neutral-active hyaluronidase glycoprotein (sHASEGP), such as human soluble PH-20 hyaluronidase glycoproteins such as rHuPH20 Annex (HYLENEX) ®, Baxter International and Inc. including (Baxter International, Inc.)). Certain exemplary sHASEGPs and methods of use (including rHuPH20) are described in U.S. Patent Publication Nos. 2005/0260186 and 2006/0104968. In one aspect, the sHASEGP is combined with one or more additional glycosaminoglycanases, such as a chondroitinase.

Exemplary lyophilized antibody preparations are described in U.S. Patent No. 6,267,958. Aqueous antibody formulations include those described in U.S. Patent Nos. 6,171,586 and WO2006 / 044908, and the latter formulations include histidine-acetate buffer.

In addition, the formulations herein may contain more than one active ingredient required for the particular indication being treated, preferably those that have complementary activities that do not affect each other. These active ingredients are suitably present in combination in amounts effective for their intended purpose.

The active ingredient can be incorporated into the microcapsules prepared by coacervation techniques or interfacial polymerization in, for example, colloidal drug delivery systems (e. G., Liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules) For example, hydroxymethylcellulose or gelatin-microcapsules and poly- (methylmethacrylate) microcapsules, respectively. Such techniques are described in Remington ' s Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980).

Sustained-release preparations can be prepared. Suitable examples of sustained-release preparations include semi-permeable matrices of solid hydrophobic polymers containing the antibody, which are in the form of shaped articles, such as films or microcapsules.

The formulations used for in vivo administration should generally be sterile. Sterilization can be easily accomplished, for example, by filtration through a sterile filtration membrane.

G. Therapeutic Methods and Compositions

Any of the anti-LRP6 antibodies provided herein may be used in therapeutic methods.

In one aspect, an anti-LRP6 antibody is provided for use as a medicament. In a further aspect, there is provided an anti-LRP6 antibody for use in treating Wnt mediated disorders, such as cancer or skeletal or bone disorders. In certain embodiments, an anti-LRP6 antibody is provided for use in a method of treatment. In certain embodiments, the invention provides an anti-LRP6 antibody for use in a method of treating such an individual, comprising administering an effective amount of an anti-LRP6 antibody to a subject having a cancer or skeletal or bone disorder. In one such embodiment, the method further comprises administering to the subject an effective amount of at least one additional therapeutic agent as described below, for example. In a further embodiment, the invention provides an anti-LRP6 antibody for use in inhibiting signaling induced by a first Wnt isoform and for enhancing signaling induced by a second Wnt isoform. In certain embodiments, the invention includes administering to a subject an effective anti-LRP6 antibody to inhibit signal transduction induced by a first Wnt isoform and enhance signal transduction induced by a second Wnt isoform , An anti-LRP6 antibody for use in a method of inhibiting signaling induced by a first Wnt isoform and enhancing signaling induced by a second Wnt isoform. An " entity " according to any of the above embodiments is preferably a human.

In a further aspect, the invention provides the use of an anti-LRP6 antibody in the manufacture or formulation of a medicament. In one embodiment, the medicament is for the treatment of Wnt mediated disorders, such as cancer or skeletal or bone disorders. In a further embodiment, the medicament is for use in a method of treating the Wnt mediated disorder, including administering an effective amount of a medicament to a subject having a Wnt mediated disorder, such as a cancer or a skeletal or bone disorder. In one such embodiment, the method further comprises administering to the subject an effective amount of at least one additional therapeutic agent as described below, for example. An " entity " according to any of the above embodiments may be a human.

In a further aspect, the present invention provides a method for treating a Wnt mediated disorder, such as cancer or skeletal or bone disorders. In one embodiment, the method comprises administering to an individual having such a Wnt mediated disorder an effective amount of an anti-LRP6 antibody. In one such embodiment, the method further comprises administering to the subject an effective amount of at least one additional therapeutic agent as described below, for example. An " entity " according to any of the above embodiments may be a human.

In one embodiment, the Wnt mediated disorder is a cancer, such as, for example, non-small cell lung cancer, breast cancer, pancreatic cancer, ovarian cancer, kidney cancer or prostate cancer. In another embodiment, the Wnt mediated disorder is a skeletal or bone disorder, such as, for example, osteoporosis, osteoarthritis, fracture or bone lesion.

One embodiment binds to LRP6 and binds to LRP6 and an antibody that inhibits signaling induced by Wnt isoforms selected from the group consisting of Wnt3 and Wnt3a and binds to Wnt 1, 2, 2b, 6, 8a, 9a, 9b and 10b Comprising administering to an individual having an cancer an effective amount of an antibody that inhibits signal transduction induced by a Wnt isoform selected from the group consisting of: &lt; RTI ID = 0.0 &gt; Another embodiment binds to LRP6 and binds to LRP6 and an antibody that inhibits Wnt3 and Wnt3a-induced signal transduction and induces signal transduction induced by Wnt 1, 2, 2b, 6, 8a, 9a, 9b and 10b Comprising administering to an individual having cancer an effective amount of an antibody that inhibits the activity of the antibody. Another embodiment is an antibody that binds to LRP6 and binds to LRP6 and an antibody that inhibits Wnt3 and Wnt3a-induced signal transduction and that binds to Wnt 1, 2, 2b, 4, 6, 7a, 7b, 8a, 9a, 9b, Comprising administering to an individual having an cancer an effective amount of an antibody that inhibits signal transduction induced by a &lt; RTI ID = 0.0 &gt; 10b. &Lt; / RTI &gt;

In a further aspect, the invention encompasses administering an anti-LRP6 antibody and a Wnt isoform enhancing signaling induced by an effective amount of Wnt isoforms to an individual to enhance Wnt signaling induced by Wnt isoforms , Provides a method of enhancing Wnt signaling induced by Wnt isoforms in an individual.

In a further aspect, the invention provides a pharmaceutical formulation comprising any of the anti-LRP6 antibodies provided herein for use, for example, in any of the above methods of treatment. In one embodiment, the pharmaceutical agent comprises any of the anti-LRP6 antibodies provided herein and a pharmaceutically acceptable carrier. In another embodiment, the pharmaceutical agent comprises any of the anti-LRP6 antibodies provided herein and at least one additional therapeutic agent, e.g., as described below.

The antibodies of the present invention may be used in therapy alone or in combination with other agents. For example, an antibody of the invention can be co-administered with at least one additional therapeutic agent. In certain embodiments, the additional therapeutic agent is a chemotherapeutic agent. In another embodiment, the agent is an antibody effective in the treatment of cancer or in the treatment of skeletal or bone disorders. Such combination therapy as referred to above includes combination administration (wherein two or more therapeutic agents are included in the same or separate agent), and individual administration, wherein administration of the antibody of the present invention comprises administration of an additional therapeutic agent and / May be performed before, concurrently with, and / or after administration of the pharmaceutical composition. The antibodies of the present invention may also be used in combination with radiation therapy.

The antibody (and any additional therapeutic agent) of the present invention may be administered by any suitable means including parenteral, intratracheal, and intranasal, and may be administered, if desired, in a lesion for local therapy. Parenteral infusions include intramuscular, intravenous, intraarterial, intraperitoneal or subcutaneous administration. Administration may be by injection, such as intravenous or subcutaneous injection, depending on any suitable route, for example partly whether the administration is short-term or long-term. Various dosing regimens, including single or multiple dosing at various times, are contemplated herein for bolus administration and pulse injection.

The antibodies of the invention will be formulated, dosed and administered in a manner consistent with good medical practice. Factors to be considered in this context include the particular disorder being treated, the particular mammal being treated, the clinical condition of the individual patient, the cause of the disorder, the delivery site of the agent, the method of administration, the schedule of administration, and other factors known to the medical practitioner. Although not necessarily so, optionally, the antibody is formulated with one or more agents currently used to prevent or treat the disorder. The effective amount of such other agents will depend on the amount of antibody present in the formulation, the type of disorder or treatment, and other factors discussed above. It is generally used by the same dosage and route of administration as described above, or by about 1 to 99% of the dosages described herein, or by any dose and route determined to be experimentally / clinically appropriate .

(For use alone or in combination with one or more other therapeutic agents) for the prevention or treatment of disease will depend on the type of disease to be treated, the type of antibody, the severity and course of the disease, Whether it is administered for prophylactic or therapeutic purposes, the prior therapy, the patient's clinical history and response to the antibody, and the judgment of the clinician. The antibody is suitably administered to the patient at one time or over a series of treatments. Depending on the type and severity of the disease, for example, from about 1 μg / kg to 15 mg / kg (eg, from 0.1 mg / kg to 10 mg / kg), whether by one or more individual doses or by continuous infusion / kg) of the antibody may be an initial candidate dose for administration to a patient. One typical daily dosage may range from about 1 [mu] g / kg to 100 mg / kg or more, depending on the factors mentioned above. For repeated administrations over several days, treatment generally continues until the disease symptoms are inhibited to a desirable level, depending on the condition. An exemplary antibody dose will range from about 0.05 mg / kg to about 10 mg / kg. Thus, a dose of at least about 0.5 mg / kg, 2.0 mg / kg, 4.0 mg / kg or 10 mg / kg (or any combination thereof) may be administered to a patient. Such a dose may be administered intermittently, e. G. Every week or every three weeks (e. G., From about 2 to about 20, or for example about 6 doses of the antibody administered to the patient) . After administration of a higher initial loading dose, one or more lower doses may be administered. The progress of such therapy is readily monitored by conventional techniques and assays.

It is understood that any of the above agents or therapeutic methods may be performed using the immunoconjugates of the invention in place of or in addition to the anti-LRP6 antibody.

H. Manufactured goods

In another aspect of the invention there is provided an article of manufacture containing a substance useful for the treatment, prevention and / or diagnosis of the disorders described above. The article of manufacture comprises a container, and a label or package insert on or in contact with the container. Suitable containers include, for example, bottles, vials, syringes, IV solution bags and the like. A container may be formed from a variety of materials, such as glass or plastic. The container holds the composition itself or holds the composition in combination with another composition effective for the treatment, prevention and / or diagnosis of a condition, and may have a sterile access port (e.g., the container may be a hypodermic needle It may be an intravenous solution or vial having a piercing cap). At least one activator in the composition is an antibody of the invention. The label or package insert indicates that the composition is used to treat the selected condition. The articles of manufacture also include (a) a first container containing the composition therein, the composition comprising an antibody of the invention; And (b) a second container containing the composition therein, the composition comprising an additional cytotoxic agent or other therapeutic agent. An article of manufacture in this embodiment of the invention may further comprise a package insert indicating that the composition can be used to treat a particular condition. Alternatively or additionally, the article of manufacture may further comprise a second (or third) container comprising a pharmaceutically acceptable buffer such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution and dextrose solution . This may further include other materials desirable from a commercial and user standpoint, such as other buffers, diluents, filters, needles and syringes.

It is understood that any such article of manufacture may comprise an immunoconjugate of the invention in place of or in addition to the anti-LRP6 antibody.

III. Example

The following are examples of the methods and compositions of the present invention. It is understood that various other embodiments may be practiced in the general description provided above.

Example 1

Experimental Procedure

Cell culture and cell assay

Cell lines EKVX and M14 were grown in RPMI-1640 medium supplemented with 10% fetal bovine serum and 2 mM glutamine; JHH-1 cells were grown in Williams &apos; s medium E with the same supplement. All other cell lines were obtained from the American Type Culture Collection (ATCC) and maintained as suggested.

Cells were transfected in a 24-well plate with FuGENE 6 transfection reagent (Roche) according to the manufacturer's recommendations. For a luciferase reporter assay, a mixture of expression plasmid DNA was transfected: 7.5 ng TOPglow (Upstate) or TOPbrite (Zhang et al., 2009) Luciferase Wnt reporter, 0.5 ng pRL-SV40 Renilla luciferase (Promega), and 1 ng LEF1. Cells were treated with antibody for 16-20 hours starting 24 h after transfection. Wnt3a protein (purified according to X, or purchased from R & D Systems) was added to the cells starting 1 hour after the start of antibody treatment. Cells were harvested in 150 ul of lysis buffer (De Almeida et al., 2007) and luminescence was detected using a Dual-Glo luciferase system (Promega) and an Envision Multi- (PerkinElmer) for 30-50 ul of lysate. Firefly luciferase levels were normalized to renilla luciferase levels for transfection efficiency and normalized relative to levels in cells where relative luciferase units (RLU) were not stimulated with Wnt3a.

Stably integrated HEK293 and Hs578T cell lines with Top Bright reporters were selected for hygromycin resistance. Expression of Wnt luciferase reporter was assessed using HEK293 cells in the presence of stably integrated SV40-driven Renilla luciferase or Hs578T cells on the basis of MultiTox-Fluor cell viability assay (Promega) Lt; / RTI &gt;

Whole-length Wnt1 or Wnt3a was cloned upstream of the full-length FZD4, FZD5 or LRP6 in the pRK5 expression vector to construct a Wnt chimeric construct. A 24-amino acid linker (GGGSGGGT) 3 was inserted between the Wnt and FZD or LRP6 sequences (Cong et al., 2004).

The 1-arm YW211.31 antibody variant is shown in Fig. The YW211.31.62 heavy chain and the light chain were co-expressed with the truncated Fc domain using knovs-into-holes technique in E. coli (Ridgway, JBB et al. Protein Engineering 9: 617-621 (1996)). For antibody bridging, Fc-specific goat-anti-human IgG antibody or F (ab ') 2 fragment (Sigma-Aldrich) was incubated with 1-arm YW211.31 antibody for 1 hour, Lt; / RTI &gt;

For Western analysis, 1.2 x 10 6 HEK293 cells were seeded on 10-cm dishes and incubated with 10 μg / ml antibody or X μg / ml DKK1 (R & D Systems) or Fzd8CRD-Fc (DeAlmeida et al. , 2007) After treatment with protein for 1 hour, 0.2 ug / ml Wnt3a protein was added for an additional 1 hour. Cells were washed twice with cold PBS and lysed in 0.5 ml lysis buffer on ice. 20 μg of protein was separated by electrophoresis on denaturing SDS-polyacrylamide gel (4-12%), transferred to a nitrocellulose membrane and incubated with phospho- and total LRP6 (Cell Signaling Technology), β - catenin (BD Transduction Laboratories), beta-actin and GAPDH. Proteins were visualized using an infrared-labeled secondary antibody (Rockland Immunochemicals) and an Odyssey imager (LI-COR).

For quantitative real-time PCR (qPCR) expression analysis, RNA was isolated from the cells using an RNeasy kit (QIAGEN) and run on a 7900 HT high-speed real-time PCR system (Applied Biosystems) 1-step RT-PCR Master Mix Reagent Kit (Applied Biosystems). Relative RNA levels were calculated using the △ ΔCt method and normalized for human GAPDH or mouse Rpl19 RNA levels in the same sample and additionally for samples from cells without Wnt3a, antibody or other protein (NA) Normalized. The primers and probe sets are as follows (listed as 5 'to 3' for forward primer, reverse primer, and probe sequence, respectively):

Figure 112012086169669-pct00003

Primers and probes used for human APCDD1, AXIN2, GAD1, LEFTY2 and SAX1, and mice Rpl19 and Axin2 have been described previously (De Almeida, et al. (2007); Liu et al. .

GAPDH primers and probes were purchased from Applied Biosystems. For reporter gene and qPCR assays, all figures represent the mean and standard deviation of three or four test replicates.

LRP6 antibody screening and affinity maturation

A human LRP6 cDNA fragment encoding region E1-E2 (amino acid A19-R644 of SEQ ID NO: 29) and E3-E4 (amino acid V629-G1244 of SEQ ID NO: 29) was cloned into the human IgG Fc region as the HSV signal sequence and protein tag -E2-fc); SEQ ID NO: 31 (E3-E4-fc)). The LRP6.E1-E2-Fc and LRP6.E3-E4-Fc proteins were expressed in CHO cells by transient transfection and purified by protein A / G affinity chromatography. In addition, the human synthetic Fab phage display library was screened using the LRP6.E1-E2-Fc and LRP6.E3-E4-Fc proteins separately. After selection on immobilized LRP6 protein, the phage clone was isolated and confirmed by phage ELISA for binding to the LRP6-Fc fusion protein fragment (and not the Fc protein). The phage Fab clones were then reformatted for expression as human IgGl monoclonal antibodies. Twenty-two characteristic antibody heavy chain clones for LRP6.E1-E2-Fc and 22 clones for LRP6.E3-E4-Fc were transfected and transiently expressed in HEK293 cells with a common Herceptin-derived human kappa light chain And the IgG protein was purified by affinity chromatography. A continuous large scale antibody preparation was produced by transient transfection in CHO cells.

The YW211.31 antibody was affinity matured using the His-tagged LRP6.E3-E4 protein. Three different combinations of CDR loops (H1 / L3, H2 / L3 and H3 / L3) were targeted for randomization in individual libraries by soft-randomized selected residues. In addition, L1 / L2 / L3 CDR combinations were targeted for hard randomization. In the first round of selection, the phage from the randomization library was selected as the fixed LRP6.E3-4-His protein, then the concentration of LRP6.E3-4-His was reduced to 5 rounds , And a 100-fold excess of LRP6.E3-4-Fc protein was added to deplete the antibody with a faster dissociation rate. Eleven phage clones were purified and all showed improved affinity for LRP3.E3-E4 as determined by phage competition ELISA. The sequence of these clones showed 1 to 6 amino acid changes in CDR-H1, CDR-H3 and CDR-L3. The dissociation rate constants of the purified antibodies were assessed by surface plasmon resonance analysis using a BIAcore instrument.

Biological layer interference method LRP6 protein binding assay

Biological layer interferometry was performed as previously described (Bouris et al., 2010). Briefly, biotinylated His-tagged LRP6 protein was purified from baculovirus-infected insect cells using the AviTag system (GeneCopoeia). Binding kinetics were measured on an Octet RED system (ForteBio) using a streptavidin high binding FA biosensor loaded with 20 μg / ml LRP6 protein. Carrier-free purified human Wnt3a and mouse Wnt9b were obtained from R & D Systems, and the purified DKK1 protein was produced as previously described (Bourish et al., 2010).

Tumor and bone research

Tumors from MMTV-Wnt1 transgenic mice were subcultured in mammary fat pads of C57BL / 6 mice, dissociated mechanically and enzymatically and replated in Matrigel and Hank Balanced Salt Solution (HBSS) And injected into the breast fat pad of a thymus NCr nude mouse (Taconic). Treatment was initiated when the tumor volume reached 250-800 mm &lt; 3 & gt ;. In each treatment group, 10 mice were dosed with 30 mg / kg of antibody or protein intraperitoneally (IP) every other day. Tumor volume was analyzed using caliper measurements.

Ntera-2 xenograft tumor growth and in vivo studies were performed as previously described (De Almeida et al., 2007). Briefly, NU / NU athymic nude mice (Charles River) were subcutaneously injected with 10-20 million Ntera-2 cells per mouse (in 50% matrigel in HBSS) and a mean tumor volume of 535-595 mm 3 , Animals were divided into 4 or 5 animal groups and injected with a single IP dose of 100 mg / kg antibody or 30 mg / kg Fzd8CRD-Fc protein. Tumor and blood serum samples were collected 16 hours after treatment. Tumors were homogenized using a TissueLyser system (quiagen) and RNA was extracted using an RNeasy kit (quiagen).

Both were harvested and cultured as described in Mohammad et al., 2008. Briefly, the two openings were dissected from two-day-old mouse pups, cut in half, and separated from the dura, blood vessels and scalp. Both openings were cultured in tissue culture plates for 1 day in 0.1% bovine serum albumin and BGJb medium supplemented with 100 U / ml penicillin and streptomycin, respectively, and then treated with 10 μg / ml antibody or protein for 7 days. The bone was incubated at 37 ° C in a humidified atmosphere of 5% CO 2 . Two mice were imaged with a μCT 40 (SCANCO Medical, Basserdorf, Switzerland) x-ray Micro-CT system. Micro-CT data were obtained with the following parameters: x-ray tube energy level = 45 kV, current = 177 μA, integration time = 300 msec, 2000 projection. Axis images were obtained at isotropic resolution of 6 [mu] m. A hydroxyapatite (HA) model was used for calibration. Micro-CT scans were analyzed with Analyze (AnalyzeDirect Inc., Lenexa, KY). For each sample, a maximum-intensity projection in the cross-section and a three-dimensional surface perspective were generated. The parietal boundary was manually drawn using a tracing tool to divide the two parts. Within this region, the sample volume and mean bone mineral density (BMD) were calculated. A threshold of 0.3 gm-HA / cm 3 was applied to the area to calculate the mean BMD of only calcified tissue within the area. We also calculated the percentage of calcification volume of the two limbs by dividing the number of calcified voxels by the total voxels for the two limbs using thresholds. The following parameters were analyzed for each sample: the two government volumes, the BMD of the two calcified voxels of the two governments, and the percentage of the two calcifications. The difference between the groups was considered significant if the p-value was less than 0.05 by the Dunnett test.

All experiments using mice were performed according to the guidelines of the SenenTech Animal Experimental Ethics Committee.

Example 2

Isolation of Wnt antagonist and enhanced LRP6 monoclonal antibody

To develop candidate therapeutic molecules for manipulating Wnt signaling, antibodies were generated that could inhibit or enhance Wnt3a protein-induced signal transduction. The recombinant LRP6.E1-E2-Fc (SEQ ID NO: 30) and LRP6.E3-E4-Fc (SEQ ID NO: 31) proteins were used to screen for a human synthetic Fab phage display library and the binding of isolated phage clones to LRP6 by ELISA Respectively. Twenty-four distinct antibody heavy chain clones for LRP6.E1-E2 and 22 clones for LRP6.E3-E4 were isolated, reformatted and expressed as human IgG1 antibodies. Six LRP6.E3-E4 antibodies inhibited the Wnt luciferase reporter activity in HEK293 cells induced with 0.1 mg / ml purified Wnt3a in a concentration-dependent manner (Fig. 1a. The error bars of all graphs represent the standard deviation of at least three repeated samples. These antibodies were named YW211.03, YW211.08, YW211.11, YW211.12, YW211.31 and YW211.33. None of the LRP6.E1-E2 antibodies showed such inhibition. YW211.31 antibody which recognizes the domain LRP6.E3-E4 was the most strongly to inhibit the signal transfer to the IC 50 of about 1 ug / ml (or 6 nM) in Wnt3a- stimulated HEK293 cells. The YW211.31 antibody inhibited Wnt3a-induced LRP6 phosphorylation and beta -catenin protein stabilization without affecting the level of LRP6 protein similarly to the purified Fzd8CRD and DKK1 proteins (Fig. 1b, unstimulated or induced with Wnt3a, Western analysis of HEK293 cells treated with the indicated LRP6 antibody or purified protein (b-actin and GAPDH protein levels are shown as sample load controls). RNAi experiments have demonstrated that only low molecular weight bands recognized by the b-catechin polyclonal antibody represent the b-catenin protein. The YW211.31 antibody can also antagonize mouse Lrp6 function, in part because it inhibits Wnt3a-induced reporter activity in mouse NIH / 3T3 cells and beta -catenin protein stabilization in mouse L cells.

The YW211.31 antibody has a binding affinity of about 2 nM by surface plasmon resonance (SPR) and 0.6 nM by Scatchard analysis. To improve the affinity and potential efficacy of the YW211.31 antibody, the clones were affinity-matured using a CDR combinatorial library in which His-tagged LRP6 E3-E4 protein and selected CDR residues were targeted for randomization. Four phage clones, YW211.31.11, YW211.31.11, 35, YW211.31.57 and YW211.31.62, which exhibited the most improved affinity by phage competition ELISA, were reformatted and expressed in whole human IgG. The dissociation rate constants of all four affinity-matured IgGs were reduced, improving the affinity for the two most potent antibodies, YW211.31.57 and YW211.31.62 (KD 0.27 and 0.17 nM, respectively). YW211.31.57 and YW211.31.62 also exhibit improved efficacy in inhibiting signaling with an IC50 value of approximately 0.1 μg / ml (0.6 nM) in Wnt3a-stimulated HEK293 cells.

No antibodies isolated on the screen activated signal transduction in HEK293 cells in the absence of stimulation with the exogenous Wnt3a protein, but five LRP6 E1-E2 and two E3-E4 antibodies inhibited Wnt3a-induced signal transduction by at least 2- The ship was fortified. In mouse NIH / 3T3 cells, the YW210.09, E1-E2 antibody also enhanced Wnt3a-induced signaling at least 1.5-fold, indicating that it also recognizes mouse LRP6. In HEK293 cells, the degree of enhancement of Wnt3a-induced signaling by the YW210.09 antibody is proportional to Wnt3a concentration (Fig. 1c). The YW210.09 antibody interacts with the human LRP6 E1-E2 protein with a KD of 5 nm as determined by SPR analysis. ELISA tests indicate that all antagonists and enhancing antibodies specifically bind to the LRP6 protein fragment used for their isolation and none recognize both E1-E2 and E3-E4. FACS analysis indicates that the soluble LRP6 E1-E4 protein efficiently and completely blocks binding of YW211.31.57 and YW210.09 to HEK293 cells, and these antibodies do not recognize other cell surface proteins.

Example 3

Self-secretion Wnt  For signaling LRP6 Monoclonal  Effect of Antibody

The ability of the LRP6 antibody to antagonize or enhance intrinsic or autocrine, Wnt signaling was determined using a variety of tumor cell lines (Bafico et al., 2004; DeAlmeida et al., 2007; Akiri et al. 2009]. In teratocarcinoma cell lines PA-1 and NTERA-2, the YW211.31 antibody inhibits reporter activity induced by autocrine Wnt signaling with an effect similar to that observed in exogenous Wnt3a (Fig. 2a shows that the luciferase reporter Showing concentration-dependent inhibition and enhancement of autocrine Wnt signaling in PA-I adenocarcinoma cells treated or infected, individually or in combination with LRP6 antibodies or treated with Fzd8CRD-Fc protein (positive control).

In PA1 cells, inhibition of Wnt signaling by the YW211.31 antibody is also observed in the expression of endogenous Wnt target genes (Fig. 2B). Fig. 2b shows the results of treatment with 10 mg / ml YW211.31 antibody, anti-gD monoclonal antibody as a negative control or PA-1 treated with Fzd8CRD-Fc protein as a positive control sample, treated or untreated with 0.3 mg / ml Wnt3a protein The results of qPCR expression analysis of Wnt-induced genes SAX1 and GADl and Wnt-inhibited gene LEFTY2 in cells are shown, which are further normalized for samples from cells without (Wnt3a) added Wnt3a.

Antibodies partially inhibit the expression of SAX1, GAD1 and APCDD1, which are either induced by the exogenous Wnt3a protein or maintained by endogenous, autocrine Wnt signaling. In contrast, inhibition of LEFTY2 expression by Wnt3a protein or autocrine Wnt signaling is resolved by the YW211.31 antibody. Unlike YW211.31, the YW210.09 antibody enhances both Wnt3a-induced and autocrine Wnt signaling in PA-1 and NTERA-2 cell lines by reporter gene assays (Fig. 2a). The inhibition of Wnt signaling by the YW211.31.57 antibody increases progressively with increasing antibody concentration, whereas the enhancement by YW210.09 and other antibodies may decrease at high antibody concentrations in some cell types, such as PA-1 cells have. This suggests that high antibody concentrations prefer the unicellular interaction and that receptor LRP6 dimerization is required for enrichment because it limits the cross-linking of LRP6 molecules. Treatment of PA-1 or NTERA-2 cells with a combination of both YW211.31.57 and YW210.09 antibodies antagonizes both Wnt3a-induced and autocrine Wnt signaling similar to the effect of YW211.31.57 alone.

Cell lines expressing relatively high expression of Axin2 mRNA or phospho-LRP5 / 6 were transfected with Wnt signaling by Fzd8CRD-Fc protein in Wnt luciferase reporter gene assays to identify additional cell lines expressing autocrine Wnt signaling &Lt; / RTI &gt; NSCLC cells NCI-H23 and NCI-H2O30 and 9 cell lines, including soft tissue sarcoma SW872 and HT-1080 (previously reported to have endogenous Wnt signaling based on assays using other Wnt antagonists), Fzd8CRD (Wu et al., 2008; Akiri et al., 2009; Nguyen et al., 2009). Figure 3 shows a summary of the data analyzed by one-way ANOVA of variance (p-value &lt; 0.01). The assays were performed using 10 mg / ml of antibody except for NCI-H358 and HT-1080 cells treated with 1 mg / ml YW211.31.57 or YW210.09 antibody, respectively, to increase the enhancing effect.

Wnt signaling was also induced by the endogenous Wnt3a protein in all 9 cell lines, and the YW211.31.57 antibody inhibited this response to Wnt3a (Figures 3, 4a, 4d and 4f). Surprisingly, while YW211.31.57 antibody enhanced self-secreted Wnt signaling in all nine of these cell lines, YW210.09 enhanced self-secreted Wnt signaling in five cell lines and inhibited it in three cell lines , 4a-4c, 4e and 4f). This mutual activity of the YW211.31.57 antibody against autocrine and Wnt3a-induced signaling was observed not only in the case of using luciferase reporters, but also in the expression of endogenous Wnt target genes such as Axin2 in the six cell lines tested 4a, 4b and 4c). In EKVX and breast carcinoma Hs578T cell lines, an increase in Wnt signaling by the YW211.31 antibody has been shown to be dependent on autocrine Wnt (s) by demonstrating that this increase is blocked by the Fzd8CRD-Fc protein (Figure 4g) . Enhancement of autocrine Wnt signaling in EKVX and Hs578T cells is also observed in antibody antagonists of five other Wnt3a-induced signal transduction pathways identified on the screen.

In Figure 4A, qPCR expression analysis of AXIN2 mRNA in HT-1080, EKVX, NCI-H358 and Hs578T showed that YW211.31.57 (25 mg / ml) antibody enhanced self-secretion (NA) Wnt signaling and Wnt3a / ml), whereas Fzd8CRD-Fc (25 mg / ml) antagonizes both autocrine (NA) and Wn3a-induced signal transduction. In Figures 4b and 4c, the expression of the Wnt-induced gene in NCI-H23 (B) and M14 (C) cells is enhanced by YW211.31.57 and is antagonized by the YW210.09 antibody (30 mg / ml) . Treatment with Wnt3a (0.2 mg / ml) and Fzd8CRD-Fc (30 mg / ml) was presented as a positive control for the enhancement and inhibition of autocrine Wnt signaling, respectively, and CD4-Fc protein (B) or anti- C) was used as a negative control (30 mg / ml). In the case of M14 cells (C), AXIN2 and SP5 expression is enhanced more strongly than APCDD1 and ZNRF3 expression by Wnt3a protein or YW211.31.57 antibody. Figures 4d and 4e show that, in Hs578T cells stably integrated with Wnt luciferase reporter, the YW211.31.57 antibody inhibited the concentration-dependent inhibition (D) of Wnt3a-stimulated signaling and the enhancement (E) of autocrine Wnt signaling , Whereas the Fzd8CRD-Fc protein inhibits signal transduction under the presence or absence (NA) of 0.1 mg / ml Wnt3a stimulation and the YW210.09 antibody enhances this signal transduction. RNAi experiments indicate that at least 41% of the Wnt3a-induced signaling in Hs578T cells is dependent on LRP5 expression and that signaling is expected to be inhibited by the Fzd8CRD-Fc protein rather than the YW211.31.57 antibody. In these experiments, it was independently verified that SV40-driven luciferase was not transfected for normalization and instead antibody and protein treatment had no significant effect on the survival rate of this cell line. Figures 4f and 4g show that EKVX cells transfected with the Wnt luciferase reporter also exhibit potentiation of autocrine Wnt signaling by YW211.31.57 antibody and antagonism of Wnt3a-induced signal transduction. Antibody-mediated enhancement of autocrine Wnt signaling is inhibited by the 5 mg / ml Fzd8CRD-Fc protein.

Example 4

Mutual activity of LRP6 antibody on different Wnt isoforms

The YW211.31 antibody inhibits exogenous Wnt3a protein-induced signal transduction in all cell lines, but it can inhibit or enhance autocrine Wnt signaling in a cell-strain-dependent manner, Suggesting that the form specifies the activity of the antibody. Thus, the activity of antibodies to signaling induced by exogenous expression of Wnt3a and other Wnt isoforms was determined. Wnt signal transduction induced by transfection of Wnt3a in HEK293 or Hs578T cells is inhibited by the YW211.31.57 antibody with an efficacy similar to inhibition of signaling induced by Wnt3a protein treatment. Surprisingly, signal transduction by Wnt1 expression in both cell lines is enhanced by the YW211.31.57 antibody. Both Wnt1 and Wnt3a signaling are inhibited by the Fzd8CRD-Fc protein as expected. Enhancement of Wnt1 signaling was also observed in other Wnt3a antagonist antibodies identified on the screen. The YW210.09 antibody also exhibited anti-Wnt3a- and Wnt1-induced signaling activity, interacting with YW211.31.57 activity, that is, enhancement of Wnt3a and inhibition of Wnt1 signaling. The YW210.09 antibody also inhibited Wnt1 signaling in tumor cells grown in cultures from MMTV-Wnt1 mouse tumors, as observed by expression of the Wnt target genes Axin2 and Mmp7, which were similarly reduced to Fzd8CRD-Fc protein treatment . In MMTV-Wnt1 cells, the YW211.31.57 antibody failed to enhance Wnt1 signaling, presumably because Wnt1 signaling is already maximal in these cells.

Since the YW211.31.57 and YW210.09 antibodies have been shown to have a mutual activity for Wnt signaling initiated by Wnt3a and Wnt1, this assay also shows that 19 luciferase reporters inducing more than 2-fold luciferase reporter in HEK293 cells RTI ID = 0.0 &gt; Wnt &lt; / RTI &gt; Figure 5 provides a summary of data from assays performed using 10 [mu] g / ml of antibody, 10 [mu] g / ml Fzd8CRD. Only Wnt3a and Wnt3 activity is inhibited by the YW211.31.57 antibody, both enhanced by YW210.09. Seven Wnt isoforms other than Wnt1 are enhanced by YW211.31.57 and inhibited by YW210.09. The third class of Wnt isoforms (Wnt7a, 7b and 10a) show signaling activity enhanced by at least YW211.31.57 without being inhibited by either antibody. In Hs578T cells transfected with different Wnt isoforms, antibodies represent most of these same activities. In particular, YW211.31.57 inhibits Wnt3 and Wnt3a and enhances at least two all other 11 Wnt isoforms that induce luciferase reporter. YW210.09 also inhibits Wnt3 and Wnt3a in Hs578T cells as well as five of the seven Wnt isoforms that are antagonized in HEK293 cells and can be tested in Hs578T cells. Two other Wnt isoforms of this class, Wnt8a and Wnt9b, are not affected by the YW210.09 antibody in Hs578T cells. RNAi experiments demonstrate that Wnt3a signaling in Hs578T is transduced by both LRP6 and LRP5, but not in HEK293 cells, so Wnt8a and Wnt9b can mainly signal through LRP5 in Hs578T cells. As in HEK293 cells, the third class of Wnt isoforms was not inhibited by either antibody in Hs587T cells and we were able to add Wnt4 (which did not induce signal transduction in HEK293 cells) to this class. Only the activity of Wnt7b in this class behaves differently in that the YW210.09 antibody enhances its signaling in Hs578T but not in HEK293 cells. In contrast to the Wnt isotype-specific activity of the YW211.31.57 and YW210.09 antibodies, the Fzd8CRD-Fc protein is able to strongly inhibit the activity of all Wnt except for Wnt6 and Wnt9b in HEK293 cells. In Hs578T cells, autocrine Wnt signaling is enhanced by both YW211.31.57 and YW210.09 antibodies, such as signal transduction induced by the expression of Wnt4, Wnt7a and Wnt7b. Thus, these three Wnt isoforms are candidates for driving autocrine signal transduction in Hs578T cells.

Several siRNAs for Wnt7b inhibited autocrine signaling in Hs578T cells, but not other Wnt isoforms, confirming specific Wnt proteins that mediate signal transduction. Since autocrine Wnt signaling in PA-1 cells is inhibited by the YW211.31.57 antibody and enhanced by YW210.09, Wnt3 or Wnt3a will probably activate endogenous signaling in these cells. Indeed, siRNA for Wnt3 inhibited autocrine Wnt signaling in PA-1 cells, but not Wnt3a. In NCI-H23 NSCLC cells and M14 melanoma cells, enhancement of autocrine Wnt signaling by YW211.31 antibody and antagonism by YW210.09 inhibited Wnt2 RNAi, which inhibits endogenous signaling in NCI-H23 cells, and Lt; RTI ID = 0.0 &gt; Wnt1 &lt; / RTI &gt; expression in M14 cells. Using multiple siRNAs, we found that Wnt2 expression in NCI-H23 cells and Wnt1 expression in M14 cells required autocrine Wnt signaling.

Because all antibodies isolated from the screen of Example 2 that antagonize signaling in Wnt3a-stimulated HEK293 cells inhibit Wnt3a stimulation in all other tested cell lines and also inhibited autocrine Wnt signaling in adenocarcinoma cell lines , It was surprising that these antibodies enhanced self-secreted Wnt signaling in the other nine cell lines tested. In addition, the YW210.09 antibody enhances Wnt3a signaling in all tested cell lines and promotes autocrine Wnt signaling in seven cell lines, while inhibiting endogenous signaling in the other three cell lines. These studies show that the different Wnt isoforms (expressed in the same cell line) determine the activity of the LRP6 antibody and that the Wnt3a antagonist and the enhancing antibody also have a mutual effect on most other Wnt proteins. The study also shows that the introduction of the same cell line of different Wnt isoforms determines the activity of the LRP6 antibody and that the Wnt3a antagonist and the enhancing antibody also have a mutual effect on most other Wnt proteins. Based on their functional interaction with two LRP6 antibodies, the 14 tested Wnt isoforms can be classified into three classes: Wnt3 and Wnt3a are inhibited by YW211.31 and enhanced by YW210.09 ; Wnt 1, 2, 2b, 6, 8a, 9a, 9b and 10b are enhanced by YW211.31 and antagonized by YW210.09; And Wnt 4, 7a, 7b and 10a are enhanced by YW211.31 and inhibited by YW210.09 (Figure 5). This classification clearly does not correspond to the proposed phylogeny of the Wnt gene even though the Wnt3 / 3a subfamily is the most evolutionarily diverse (Cho et al., 2010).

Example 5

Wnt isoforms specify different activities of LRP6 antibodies

It is contemplated that different Wnt isoforms may preferentially bind to different FZD isoforms that are expressed intrinsically in various cell lines, which may account for the differential activity of the LRP6 antibodies of the invention. To investigate this possibility, chimeric proteins linked by covalent linkages of different Wnt-FZD pairs were constructed to test whether specific Wnt or FZD isoforms determine the activity of LRP6 antibody. Wnt3a or Wnt1 fused to FZD4 or FZD5 strongly activates Wnt signaling in HEK293 cells under the presumed absence of endogenous Wnt expression, whereas overexpression of FZD4 or FZD5 does not induce Wnt signaling. The YW211.31.57 antibody inhibits the signaling activity of Wnt3a fused to FZD4 or FZD5 and enhances the activity of Wnt1 fused to FZD4 or FZD5 (Fig. 6, using 10 μg / ml antibody, 10 μg / ml Fzd8CRD Providing a summary of the data of the tests performed). The YW210.09 antibody exhibits mutual activity (both suppressed) against Wnt1 chimera. Thus, the activity of the antibody is related to the Wnt isotype and not to the FZD isotype. The Fzd8CRD-Fc protein does not affect signaling induced by any of the four Wnt-FZD chimeras, consistent with the independently functioning chimeras of the FZD-binding site of Wnt.

Expression of chimeras fusing Wnt1 or Wnt3a into LRP6 induces Wnt signaling much more potently than overexpression of LRP6. The YW211.31.57 and YW210.09 antibodies can not inhibit this induction, consistent with the hypothesis that the inhibitory function of the antibody is dependent on blocking Wnt binding to LRP6 (Figure 6).

This study confirms that the isotype of Wnt, rather than FZD, determines the activity of the antibody. The chimeric protein function of Wnt isoforms with LRP6, but not FZD, is not sensitive to inhibition by the LRP6 antibody suggesting that antagonism can be mediated by blocking ligand-secreting receptor interactions. This is confirmed by in vitro binding studies in both Wnt3a and YW210.09 antibodies that competitively bind within the E3-E4 region of LRP6, and Wnt9b and YW211.31 antibodies that compete for binding in the E1-E2 region. The epitopes of the two LRP6 antibodies each define a binding site to a different class of Wnt isoforms, one within E1-E2 and one within the E3-E4 domain. At least a third Wnt binding site is expected for an isotype that is not inhibited by antibodies or combinations thereof, and it is likely that the four repeat domains each bind to a different subset of Wnt isoforms. Such module organization may allow different Wnt structural diversity and their binding sites to accommodate differential regulation by Wnt-binding and co-receptor binding antagonists such as SFRP and DKK protein isoforms, respectively.

Example 6

Antibody-mediated enhancement of Wnt signaling is associated with LRP6 dimerization

Enhancement of autocrine Wnt signaling by the YW211.31 antibody probably requires a binding effect through LRP6 dimerization. The monovalent Fab fragments of YW211.31 and recombinant 1-arm YW211.31 antibodies do not exhibit enhancement of autocrine Wnt signaling in these cells at concentrations that inhibit Wnt3a-induced signaling in EKVX and Hs578T cell lines. In contrast, the YW211.31 Fab fragment and the 1-arm mAb inhibit both autologous Wnt and Wnt3a-induced signal transduction in the PA-I adenocarcinoma cell line with similar potency against intact IgG antibodies. In order to test whether cross-linking of the 1-arm YW211.31 antibody restored the Wnt-enhancing function of whole IgG molecules, self-secreted Wnt and Wnt2-induced signaling enhanced by YW211.31.57 antibody as well as Fc bridging HT-1080 soft tissue sarcoma cell lines expressing enhanced apomic antibody-induced apoptosis were used (Adams et al., 2008). The 1-arm YW211.31 antibody does not affect signal transduction induced by autocrine Wnt signal transduction or Wnt2 transfection. Under cross-linking conditions with anti-Fc antibodies that increase apomorph-mediated apoptosis, the cross-linking of the 1-arm antibody partially localizes the enhancement of both autologous Wnt and Wnt2 signaling observed in the YW211.31.57 whole antibody It was decided.

Antibody-mediated Wnt enrichment requires secondary receptor dimerization since the 1-arm and Fab antibody formats fail to enhance Wnt signaling if not cross-linked. In addition, the cell-based biochemical data presented herein indicate that Wnt binding to cross-linked LRP6 is also required for enhancing signal transduction, perhaps reflecting the need for Wnt-mediated mobilization of FZD complexes. Although a small fraction of overexpressed LRP6 can be identified as a homodimer on the cell surface and dimerization requires an extracellular domain, this contributes to Wnt-independent b-catenin signaling induced by LRP6 overexpression Is not clear (Liu et al., 2003). Depletion of the LRP6 extracellular domain also activates signal transduction in a Wnt-dependent manner, and forced extracellular dimerization of this recombinant protein by different methods can enhance or inhibit this activity (Liu et &lt; RTI ID = 0.0 &gt; al Cong et al., 2004). Both Wnt induce LRP6 aggregation and phosphorylation in the plasma membrane, which requires homologous-oligomerization of the intracellular DVL protein (Bilic et al., 2007). These large aggregates also contain Axin and GSK3 and will probably inhibit b-catenin degradation.

Example 7

Antibody-mediated enhancement of Wnt signaling by binding inhibition of Wnt antagonists

Inhibition of the activity of extracellular LRP6 antagonists such as DKK1 isoform and SOST can also enhance Wnt signaling (Niida et al., 2004). The exogenous DKK1 protein inhibits Wnt1 -induced signaling in HEK293 cells, the YW211.31.57 antibody can block this antagonism, and can even enhance signaling at high enough concentrations in the presence of the DKK1 protein (Fig. 4G ). In contrast, the YW211.31 1-arm antibody suppresses only very weakly the DKK1 antagonism of Wnt1 signaling at these same concentrations. The YW211.31.57 whole antibody effectively antagonizes DKK1 activity at all of the DKK1 concentrations tested, while the 1-arm antibody showed minimal or no effect at even low DKK1 concentrations. Strong antagonism of exogenous DKK1 activity that is observed in whole YW211.31 antibody but not in 1-arm YW211.31 antibody can contribute to Wnt-enhancing activity specific for whole antibody. Alternatively, since the DKKl protein does not completely inhibit Wnt1-induced signaling in this assay, the intact YW211.31 antibody can also simply enhance the residual signal through LRP6 dimerization.

Inhibition of DKK1 activity is probably due to the presence of residual signal transduction by Wnt coupled to secondary receptors, since antibody inhibition of DKK1 interaction with LRP6 does not necessarily confer Wnt enhancing activity and DKK1 antagonism appears to be required for LRP6 dimerization It will be mediated predominantly by strengthening.

Example 8

Wnt signaling antagonism predominates in LRP6 antibody combinations

The assay shows that Wnt3a and Wnt3 bind within the E1-E2 region of LRP6 and are inhibited from binding by the YW211.31.57 antibody. The Wnt1 isotype of the Wnt1 class is expected to bind to the E3-E4 region, and this binding is blocked by the YW210.09 antibody. Without wishing to be bound by any theory, the enhancement of Wnt signaling can occur when both the Wnt isoform and the antibody are able to bind to the same LRP6 molecule, possibly by mobilization of FZD by Wnt and LRP6 dimerization by the antibody . This model predicts that although the LRP6 dimerization may still occur, the combination of the two antibodies will inhibit signaling by one class of Wnt isoforms because the Wnt binding will be blocked by either one antibody or another . As expected, HEK293 cells inhibited signal transduction initiated by the expression of Wnt3a or Wnt1 upon simultaneous treatment with YW211.31.57 and YW210.09 antibodies (FIGS. 7 and 8a). The assay shown in Figure 8a was performed in HEK293 cells stably integrated with Wnt3a, Wnt1, or Wnt luciferase reporter transfected with expression constructs for both Wnt3a and Wntl. All antibodies and proteins were used at 10 μg / ml each. This assay was extended to three different Wnt isoforms of the Wnt1 class and the combination of the YW211.31.57 and YW210.09 antibodies was found to inhibit Wnt signaling to a similar extent as YW210.09 alone (Figure 7). When both Wnt3a and Wnt1 were expressed simultaneously, this antibody did not antagonize Wnt signaling, but the combination of the two antibodies inhibited signal transduction (FIG. 8A). One possible explanation for this result is that each antibody inhibits the binding of only one Wnt isoform and the two antibodies block both Wnt binding sites because they can bind to the LRP6 molecule simultaneously.

Four Wnt isoforms of the third class that are not antagonized by the YW211.31.57 or YW210.09 antibodies can bind to sites on LRP6 that are not blocked by any antibody, or alternatively, Lt; RTI ID = 0.0 &gt; Wnt-binding &lt; / RTI &gt; For each of these Wnt isoforms, the combination of the two antibodies also does not inhibit their signal transduction, rather it enhances or does not affect their activity, indicating that these Wnt isoforms are YW211.31.57 and YW210. Lt; RTI ID = 0.0 &gt; epitope &lt; / RTI &gt; (Figure 7).

The observed activity of the YW211.31.57 and YW210.09 antibody combinations on Wnt signaling induced by exogenous Wnt isoforms also extends to endogenous, autocrine Wnt signaling. In the teratocarcinoma cell lines PA-1 and Ntera-2, where the YW211.31.57 antibody inhibits autocrine Wnt signaling and YW210.09 enhances autocrine Wnt signaling, the antibody combination inhibits signal transduction (FIG. 2a). In Hs578T and EKVX cells, in which both antibodies enhance autocrine Wnt signaling, antibody combinations are also enhanced (FIGS. 8b and 8c).

Example 9

LRP6 antibody differentially inhibits Wnt binding to multiple sites

The mutual activity of the LRP6 antibody suggests that YW211.31.57 and YW210.09 interact with the characteristic Wnt isoform binding site on LRP6 and that Wnt binding is competed by the antagonist antibody but is permitted by the enhancing antibody. A bi-layer interferometry assay (see Example 1) that measures purified Wnt protein binding to purified and immobilized LRP6 extracellular domain protein fragments revealed that Wnt3a binds to the E3-E4 region of LRP6 (here an epitope for the YW211.31.57 antibody And Wnt9b (which is the same class as Wnt1 for antibody interaction) binds only to the E1-E2 region (where the YW210.09 antibody also binds) (Bourish et al. (2010)]). The YW211.31.57 antibody inhibited the binding of Wnt3a to the LRP6.E1-E4 protein fragment, but not YW210.09 (Fig. 9A). In contrast, YW210.09 inhibited Wnt9b binding to LRP6.E1-E4, but YW211.31.57 did not (Fig. 9b). In these assays, antibody binding to the LRP6 protein allowed equilibrium to arrive and a continuous wave shift in the interference pattern for the binding and dissociation phases of Wnt protein binding.

Antibody-mediated inhibition of Wnt binding can also be detected using smaller Wnt-binding fragments, E3-E4 for Wnt3a and E1-E2 for Wnt9b (Figures 9c and 9d). The 1-arm YW211.31 antibody can also inhibit Wnt3a binding to E3-E4 fragments. In addition, YW211.31.57 and YW210.09 can be sequenced to the LRP6.E3-E4 protein when added in competition and in any order (Fig. 9E). Competition for binding between only Wnt3a and YW211.31.57 antibodies and only between Wnt9b and YW210.09 at different sites on the LRP6 protein is associated with the inhibitory activity of each antibody on signaling by specific Wnt isoforms.

Biological interferometry assays have shown that previously purified DKKl proteins can bind both E3-E4 and E1-E2 fragments of LRP6 and that this binding can inhibit the binding of Wnt3a and Wnt9b to their respective protein domains . (Bourish et al. (2010)). Using this assay, we showed that the YW211.31.57 and YW210.09 antibodies can inhibit DKK1 binding to the LRP6.E1-E4 protein, respectively. The YW211.31.57 antibody also inhibits DKK1 binding to the LRP6.E3-E4 protein and the YW210.09 antibody blocks binding of DKK1 to the LRP6.E1-E2 fragment. Although the 1-arm YW211.31 antibody can not enhance Wnt signaling and can not significantly antagonize exogenous DKK1 activity in Wnt signaling in cells, its inhibitory activity remains intact. These results suggest that DKK1 antagonism may not contribute significantly to the antibody-mediated enhancement of Wnt signaling.

Example 10

LRP6 antibodies are active against Wnt-driven tumors and bone formation

To begin the study of the anti-tumor therapeutic efficacy of the LRP6 antibody, two models of Wnt ligand-driven tumors were treated. MMT-Wnt1 transgenic breast tumor xenografts dependent on Wnt1 expression and Ntera-2 human teratocarcinoma xenografts driven by autocrine Wnt signaling of unknown Wnt isoforms (DeAlmeida et al., 2007). Tumors were established with athymic nude mice using cells isolated from MMTV-Wnt1 transgenic mouse breast tumors and treated with antibody every other day. Rapid and sustained tumor regression was observed in the YW210.09 antibody, similar to the Fzd8CRD-Fc protein (Figure 10a). The YW211.31.57 antibody did not alter tumor growth under these conditions compared to control buffer (PBS) or anti-gD antibody treatment. Mice were dosed with 30 mg / kg of antibody or protein every other day (arrow marks) (Fig. 10a). These results are consistent with the antibody effects described above for Wnt target gene expression on MMTV-Wnt1 tumor cells treated in tissue culture.

In addition, xenograft tumors were established in athymic nude mice using Ntera-2 teratocarcinoma cells and treated with antibodies or Fzd8CRD-Fc protein. RNA extracted from tumors treated with the antibody YW211.31.57, 1-arm YW211.31, or a combination of YW211.31.57 and YW210.09 was used to express the expression of the Wnt target gene SP5 in a dose of 41 -57%, whereas Fzd8CRD-Fc protein treatment reduced SP5 expression to 8.0%. SP5 mRNA levels were normalized for GAPDH mRNA levels in the same tumor and further normalized for PBS-treated tumors. All treatments except YW210.09 show a p-value < 0.005 by ANOVA compared to PBS control. (Fig. 10B). Axin2 expression was reduced to only 56.2% by Fzd8CRD-Fc, and no significant change in Axin2 expression was detected in any of the antibody treatments. YW210.09 antibody treatment did not significantly affect the expression of SP5 or Axin2. The sera samples tested for inhibition or enhancement of Wnt3a-induced signaling in HEK293 cells confirmed that the injected antibodies and proteins retained at least some activity in vivo over the entire 16 hour exposure.

Activation or enhancement of Wnt signaling can increase bone mass by enhancing osteoblast differentiation and function, and indirectly inhibiting osteoclast differentiation (Glass et al., 2005) The activity of LRP6 antibody against mouse clavicle was examined. The two dissected open-ended explants were incubated with antibody or RANK-Fc, followed by analysis of the pit volume and density by micro-computed tomography. Using the histogram analysis of the control samples, the X-ray attenuation range for calcification (bone) and non-calcification (cartilage) tissue was determined. Treatment with the YW210.09 antibody significantly increased the mean bone mineral density (BMD) of the calcified parietal bone to 7.4%, which was similar to the 6.8% increase observed with RANK-Fc treatment to inhibit osteoclast differentiation (Fig. 10c; Hsu et al., 1999). Treatment with YW211.31.62 antibody did not significantly change calcified bony PMD. All treatments were 10 μg / ml antibody or protein for 7 days. In Fig. 10C, the data points represent two rounds of eight halves from four mice in each treatment group; The mean and standard deviation of the mean are indicated by the horizontal and vertical lines, respectively. Only YW210.09 and RANK-Fc treatments were significantly different from untreated samples by 0.01 and 0.05 p-values (<0.05 for both by t-test) by Dunnett's test, respectively.

The volume of the total bony crest bone area (calcified and non-calcified) and the proportion of calcified bone in this area were not significantly altered by antibody or RANK-Fc treatment, indicating that the YW210.09 antibody did not change mineralization Suggesting that it can be promoted.

Example 11

The LRP6 bispecific antibody acts as a Pan-Wnt inhibitor

A bispecific IgG hybrid with YW211.31.62 and YW210.09 heavy chain heterodimers was constructed using Knots-In-Hort engineering (Atwell et al., 1997). this. This LRP6 bispecific antibody produced in E. coli or HEK293 cells antagonizes Wnt3a-induced (0.1 μg / ml) signaling in HEK293 cells (Figure 11a) and tumor cell lines PA-1, M14 and CAL-51 11c). Notably, bispecific antibodies inhibit as strongly as YW211.31 and do not retain the Wnt3a- enhancing activity of YW210.09. Bispecific antibodies also inhibit autocrine Wnt signaling in all three tumor cell lines tested, while retaining the inhibitory activity of YW211. 09 antibody in PA-I cells and YW210.09 in M14 cells (Fig. 11b). Interestingly, bispecific antibodies suppress signal transduction, although YW211.31 enhances autocrine Wnt signaling in CAL-51 breast carcinoma cells and YW210.09 does not. This novel antagonistic activity is not observed in the combination of YW211.31 and YW210.09 antibodies. In this assay, PA-1 and M14 cells stably integrated with the Wnt luciferase reporter and CAL-51 cells transfected with the reporter cells were stimulated with the presence (11c) or absence (11b) of stimulation with 0.1 μg / ml Wnt3a (PBS), antibody, antibody combination, or Fzd8CRD-Fc protein (10 [mu] g / ml each).

When tested for signal transduction induced by transfection of 13 Wnt isoforms in HEK293 cells, bispecific antibodies strongly inhibited all Wnt blocked by YW211.31 or YW210.09 (Figure 12) . The assays summarized in Table 12 were performed using antibodies or proteins (10 [mu] g / ml) for signal transduction induced by transfection of expression constructs against the Wnt isotype in HEK293 or Hs578T cell lines stably integrated with Wnt luciferase reporter Effect was determined. Reporter activity was normalized to the number of cells in the transfected cells with the same expression constructs untreated with antibody or protein and further normalized to the level. Anti-gD was used as a control. The drainage-change values were calculated to be less than 0.80 for inhibition in HEK293 cells, greater than 1.30 for enrichment, less than 0.65 for inhibition in Hs578T cells and 1.30 for inhibition in Hs578T cells when they deviate from the range observed in control anti- ) Were deemed appropriate.

Similar to the combination of YW211.31 and YW210.09 antibodies, and unlike either antibody alone, the bispecific antibody blocks signaling induced by the combination of Wnt1 and Wnt3a (Figure 12). Surprisingly, bispecific antibodies also reduce signaling by the three Wnts that are not inhibited by the single or combined homodimeric antibodies. This antagonistic activity of bispecific antibodies is also observed in Hs578T cells, and it is possible to exclude the lack of effect on Wnt7a-induced signaling.

The ability of bispecific antibodies to inhibit Wnt3a-induced stabilization of the beta -catenin protein was investigated. HEK293 cells transfected with or transfected with Wnt3a were treated with YW211.31, YW210.09, or a bispecific antibody, or a control Fzd8CRD-Fc protein or anti-gD at a concentration of 5 μg / ml for 18 hours, beta -catenin protein levels and phosphorylated LRP5 / 6 levels were determined by Western blot analysis. 13a. In HEK293 cells, bispecific antibodies inhibit Wnt3a-induced stabilization of the beta -catenin protein, unlike YW210.09, similar to YW211.31 and increasing beta -catenin levels (Figure 13a). Both bispecific and YW211.31 antibodies blocked induction by Wnt3a of the high molecular weight species of phosphorylated LRP5 / 6, but the YW210.09 antibody was not. Surprisingly, YW211.31 and YW210.09 do not affect the steady state levels of the total LRP6 protein, whereas bispecific antibodies increase the LRP6 protein in the presence or absence of Wnt3a induction. In the absence of Wnt stimulation, this stabilized LRP6 could slightly increase Ser1490 phosphorylation even though the bispecific antibody did not affect Wnt reporter activity in HEK293 cells in the absence of Wnt.

The ability of bispecific antibodies to inhibit Wnt signaling in vivo was also determined. SCID-bg mice with M14 melanoma xenograft tumors were injected with 30 mg / kg LRP6 bispecific antibody, Fzd8CRD protein (positive control), or anti-gD antibody (negative control). After 16 hours of treatment, RNA was extracted and examined for expression of Wnt target gene by qPCR. mRNA levels were normalized for GAPDH mRNA levels in the same tumor and further normalized for anti-gD-treated tumors. All bispecific antibodies and Fzd8CRD treatment show a p-value < 0.001 by ANOVA compared to anti-gD control. As shown in Figure 13b, the LRP6 bispecific antibody inhibited Wnt signaling in M14 melanoma cells grown as xenograft tumors. RNA extracted from antibody treated tumors represents expression of Wnt target genes AXIN2 and APCDD1, respectively, reduced to 46-57% and 35-38% of the levels in tumors treated with control anti-gD antibody. This reduced expression level is similar to that observed in the injection of the Fzd8CRD protein, indicating that the bispecific antibody is stable and active in vivo.

Example 12

Structure of LRP6 E1-YW210.09 Fab Complex.

The crystal structure of the first β-propeller and EGF domain of LRP6 (also referred to as E1) in the complex with YW210.09 Fab was determined by molecular substitution and refined to a 1.9 Å resolution with R and Rfree of 0.175 and 0.220, respectively Respectively. The crystallographic asymmetric unit consists of one LRP6 E1 domain and one YW210.09 Fab. The interpretable electron density permits tracing of residues Ala 20 to Lys 324 for the E1 domain, residues Asp 1 to Glu 213 and Glu 1 to Lys 214 for the Fab light chain and the heavy chain respectively, except for the Fab heavy chain residues Ser 127 to Thr 131 (Overall Kabat numbering is used).

The LRP6 E1 domain is assembled into a modular framework containing a beta-propeller module and an epidermal growth factor (EGF) -like module. The β-propeller consists of six blades formed by a four-stranded anti-parallel β-sheet arranged radially with an N-terminal edge facing the center channel and a YWTD motif located on the second strand of each blade. The LRP6 E1 [beta] -propeller structure is very similar to LDLr with an rmsd of 0.83 A when superimposed on 245C-alpha atoms, despite only 36% sequence identity (Jeon, H., et al., 2001) . Most of the conserved residues are concentrated around the YWTD core motifs that form β-sheets essential for β-propeller structural integrity, but surface residues are very diverse and contribute to the functional diversity of these receptors. LRP6 uses its EGF-like domain to fix down the first and sixth blades of the propeller and maintain its mechanical strength. The EGF-like module extends from the [beta] -propeller through the 10-residue linker to the C-terminus, folds back against the lower side of the [beta] -propeller, and is docked to the surface between the third and fourth blades. The interaction between the EGF and the [beta] -propeller is broad as indicated by the large total landfill surface area of 1226 A &lt; 2 &gt; and the shape complementarity of 0.74. Leu 296, Leu 298 and Met 299 in the first beta-strand of the EGF module constitute a hydrophobic core packed into a complementary cavity of a beta -propeller surrounded by some direct or water mediated polar interaction. This feature is also observed in the LDLR structure (Jeon, H., et al., 2001; Rudenko, G., et al., 2002).

The YW210.09 Fab often recognizes the region of the upper center of the [beta] -propeller, which is a zone found to be involved in protein-protein interactions (Springer, T. A., 1998). The paratope consists of residues from five CDRs comprising three heavy chain CDRs (H1, H2, H3) and two light chain CDRs (L1 and L3). Antibodies that bind to the [beta] -propeller are filled with a total area of 1691A2 with a shape complementarity score of 0.76. The acidic patch on the upper opposing face of the? -propeller occupies approximately one third of the total area, but does not nearly overlap with the YW210 epitope. Conversely, the heavy and light chains recognize separate zones. The direct contact formed by the heavy chain CDR represents 80% of the buried area, with CDR H3 alone accounting for more than 50%. These fragments consist of 17 residues, of which residues His 98 to Lys 100c form direct contact with the [beta] -propeller. Importantly, Asn 100 of the antibody creates a pair of hydrogen bonds with Asn 185 of LRP6 to form a " handshake " interaction (Figure 16). In addition, the Val 100b and Lys 100c backbones rearrange the carbonyl groups that interact with Arg 28 of LRP6 and the two NH groups previously interacting with the acidic patch through two water molecules (Wat 1 and Wat 2) in exceptional fashion (Fig. 14). The Lys 100c side chain also neutralizes the acidic patch by hydrogen bonding with Val 70 and Ser 96 backbone carbonyl of LRP6. Arg 141 of LRP6 is anchored to the center and interacts with bridges Wat2, Asn 185 of LRP6, and Ala 100a of YW210.09. Arg 141 appears to incorporate two hydrogen bonding networks together. In addition, the Val 100b side chain is docked with the hydrophobic cavity of the central channel of the [beta] -propeller. Thus, the YW210.09 H3 sequence NAVK shows a rare combination pattern with the β-propeller E1 of LRP6. Other CDRs interact with residues in the parameters on top of the [beta] -propeller. Other residues involved in H3 binding to LRP6 include E51, D52, V70, S71, E73, L95, S96, D98 and E115. H1 and H2 contact the fifth and sixth blades, and L1 and L3 contact the sixth, first and second blades (Fig. 15). Additional LRP6 residues involved in the binding of YW210.09 to LRP6 include R29, W188, K202, P225, H226, S243 and F266. The crystal packing interaction is not directly related to the region where YW210.09 contacts the LRP6 epitope, indicating that the crystal structure should reflect how the two molecules interact in solution. The interaction between the characteristic CDR H3 NAVKN (SEQ ID NO: 49) motif and the LRP6 E1 [beta] -propeller is highly similar to the reported interactions between laminin and Nidogen (Takagi, J., et al., 2003). In both cases, a significant contact is made through the Asn hand shake described above, and the branched hydrophobic residues entering the hydrophobic cavity formed by the top of the closed-channel ß-propeller center channel in both propellers by Phe shutters. Human Dkk1 has the same motif (amino acid 40-44: NAIKN (SEQ ID NO: 50)) identical to the motif found in the CDR H3 loop of YW210.09, except for its Ile 42. This motif is strictly conserved among family members except for species and Dkk3, and represents a specific function for this motif in Dkk biology. Preserved motifs have never been previously considered (Brott, B. K., and Sokol, S. Y., 2002) and are found on the N-terminus of Dkk1, the region predicted to be disordered. In addition, this particular motif also includes two other proteins that regulate Wnt signaling through interaction with LRP5 / 6: Sclerostin (Semenov, M., et al., 2005) ) And Wise (Itasaki, N., et al., 2003). These two proteins belong to the same super-family of cysteine knock proteins (McDonald, NQ, and Hendrickson, WA, 1993) and are also identified in their loop number 2, (Lintern, KB, et al., 2009; Veverka, V., et al., 2009).

Example 13

Exemplary anti-LRP6 antibodies

The amino acid sequence of a particular anti-LRP6 antibody is provided in the sequence listing. Table 2-4 provides a description of the sequence. The alignment of the amino acids of the VH and VL domains of a particular anti-LRP6 antibody is provided in Figures 16 and 17.

Figure 112012086169669-pct00004

Figure 112012086169669-pct00005

Figure 112012086169669-pct00006

While the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it should be understood that the description and examples are not intended to limit the scope of the invention. The disclosures of all patents and scientific references cited herein are expressly incorporated herein by reference in their entirety.

references

Figure 112012086169669-pct00007
Figure 112012086169669-pct00008
Figure 112012086169669-pct00009
Figure 112012086169669-pct00010
Figure 112012086169669-pct00011
Figure 112012086169669-pct00012

                         SEQUENCE LISTING &Lt; 110 > GENENTECH, INC., Et al.   <120> ANTI-LRP6 ANTOBODIES <130> P4430R1-WO &Lt; 150 > US 61 / 394,836 <151> 2010-10-20 &Lt; 150 > US 61 / 317,137 <151> 2010-03-24 <160> 58 <170> PatentIn version 3.5 <210> 1 <211> 456 <212> PRT <213> Artificial Sequence <220> <223> Synthetic polypeptide <400> 1 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Thr Ser Tyr             20 25 30 Tyr Ile Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val         35 40 45 Ala Glu Ile Ser Pro Tyr Ser Gly Ser Thr Tyr Tyr Ala Asp Ser Val     50 55 60 Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys                 85 90 95 Ala Leu Arg Ala Arg Pro Pro Ile Arg Leu His Pro Arg Gly Ser Val             100 105 110 Met Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser         115 120 125 Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr     130 135 140 Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro 145 150 155 160 Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val                 165 170 175 His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser             180 185 190 Ser Val Val Thr Val Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile         195 200 205 Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val     210 215 220 Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala 225 230 235 240 Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro                 245 250 255 Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val             260 265 270 Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val         275 280 285 Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln     290 295 300 Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln 305 310 315 320 Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala                 325 330 335 Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro             340 345 350 Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr         355 360 365 Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser     370 375 380 Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr 385 390 395 400 Lys Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr                 405 410 415 Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe             420 425 430 Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys         435 440 445 Ser Leu Ser Leu Ser Pro Gly Lys     450 455 <210> 2 <211> 214 <212> PRT <213> Artificial Sequence <220> <223> Synthetic polypeptide <400> 2 Asp Ile Gln Met Thr Gln Ser Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Ser Thr Ala             20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile         35 40 45 Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Ser Ser Phe Ser Gly     50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Thr Thr Pro Pro                 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala             100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly         115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala     130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser                 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr             180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser         195 200 205 Phe Asn Arg Gly Glu Cys     210 <210> 3 <211> 456 <212> PRT <213> Artificial Sequence <220> <223> Synthetic polypeptide <400> 3 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Thr Ser Tyr             20 25 30 Tyr Ile Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val         35 40 45 Ala Glu Ile Ser Pro Tyr Ser Gly Ser Thr Tyr Tyr Ala Asp Ser Val     50 55 60 Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys                 85 90 95 Ala Leu Arg Ala Arg Pro Pro Ile Arg Leu Tyr Pro Arg Gly Ser Val             100 105 110 Met Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser         115 120 125 Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr     130 135 140 Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro 145 150 155 160 Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val                 165 170 175 His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser             180 185 190 Ser Val Val Thr Val Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile         195 200 205 Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val     210 215 220 Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala 225 230 235 240 Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro                 245 250 255 Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val             260 265 270 Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val         275 280 285 Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln     290 295 300 Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln 305 310 315 320 Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala                 325 330 335 Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro             340 345 350 Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr         355 360 365 Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser     370 375 380 Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr 385 390 395 400 Lys Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr                 405 410 415 Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe             420 425 430 Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys         435 440 445 Ser Leu Ser Leu Ser Pro Gly Lys     450 455 <210> 4 <211> 214 <212> PRT <213> Artificial Sequence <220> <223> Synthetic polypeptide <400> 4 Asp Ile Gln Met Thr Gln Ser Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Ser Thr Ala             20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile         35 40 45 Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Ser Ser Phe Ser Gly     50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Thr Leu Pro Thr                 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala             100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly         115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala     130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser                 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr             180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser         195 200 205 Phe Asn Arg Gly Glu Cys     210 <210> 5 <211> 456 <212> PRT <213> Artificial Sequence <220> <223> Synthetic polypeptide <400> 5 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Gly Tyr Tyr             20 25 30 Tyr Ile Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val         35 40 45 Ala Glu Ile Ser Pro Tyr Ser Gly Ser Thr Tyr Tyr Ala Asp Ser Val     50 55 60 Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys                 85 90 95 Ala Leu Arg Ala Arg Pro Pro Ile Arg Leu His Pro Arg Gly Ser Val             100 105 110 Met Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser         115 120 125 Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr     130 135 140 Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro 145 150 155 160 Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val                 165 170 175 His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser             180 185 190 Ser Val Val Thr Val Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile         195 200 205 Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val     210 215 220 Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala 225 230 235 240 Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro                 245 250 255 Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val             260 265 270 Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val         275 280 285 Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln     290 295 300 Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln 305 310 315 320 Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala                 325 330 335 Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro             340 345 350 Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr         355 360 365 Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser     370 375 380 Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr 385 390 395 400 Lys Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr                 405 410 415 Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe             420 425 430 Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys         435 440 445 Ser Leu Ser Leu Ser Pro Gly Lys     450 455 <210> 6 <211> 214 <212> PRT <213> Artificial Sequence <220> <223> Synthetic polypeptide <400> 6 Asp Ile Gln Met Thr Gln Ser Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Ser Thr Ala             20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile         35 40 45 Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Ser Ser Phe Ser Gly     50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Thr Thr Pro Pro                 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala             100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly         115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala     130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser                 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr             180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser         195 200 205 Phe Asn Arg Gly Glu Cys     210 <210> 7 <211> 456 <212> PRT <213> Artificial Sequence <220> <223> Synthetic polypeptide <400> 7 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Thr Asn Ser             20 25 30 Tyr Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val         35 40 45 Gly Trp Ile Thr Pro Tyr Gly Gly Tyr Thr Asn Tyr Ala Asp Ser Val     50 55 60 Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys                 85 90 95 Ala Arg Gly Ser Gly His Val Asn Ala Val Lys Asn Tyr Gly Tyr Val             100 105 110 Met Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser         115 120 125 Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr     130 135 140 Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro 145 150 155 160 Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val                 165 170 175 His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser             180 185 190 Ser Val Val Thr Val Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile         195 200 205 Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val     210 215 220 Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala 225 230 235 240 Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro                 245 250 255 Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val             260 265 270 Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val         275 280 285 Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln     290 295 300 Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln 305 310 315 320 Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala                 325 330 335 Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro             340 345 350 Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr         355 360 365 Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser     370 375 380 Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr 385 390 395 400 Lys Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr                 405 410 415 Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe             420 425 430 Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys         435 440 445 Ser Leu Ser Leu Ser Pro Gly Lys     450 455 <210> 8 <211> 214 <212> PRT <213> Artificial Sequence <220> <223> Synthetic polypeptide <400> 8 Asp Ile Gln Met Thr Gln Ser Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Ser Thr Ala             20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile         35 40 45 Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Ser Ser Phe Ser Gly     50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Thr Thr Pro Pro                 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala             100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly         115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala     130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser                 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr             180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser         195 200 205 Phe Asn Arg Gly Glu Cys     210 <210> 9 <211> 118 <212> PRT <213> Artificial Sequence <220> <223> Synthetic polypeptide <400> 9 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Thr Ser Tyr             20 25 30 Tyr Ile Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val         35 40 45 Ala Glu Ile Ser Pro Tyr Ser Gly Ser Thr Tyr Tyr Ala Asp Ser Val     50 55 60 Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys                 85 90 95 Ala Leu Arg Ala Arg Pro Pro Ile Arg Leu His Pro Arg Gly Ser Val             100 105 110 Met Asp Tyr Trp Gly Gln         115 <210> 10 <211> 108 <212> PRT <213> Artificial Sequence <220> <223> Synthetic polypeptide <400> 10 Asp Ile Gln Met Thr Gln Ser Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Ser Thr Ala             20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile         35 40 45 Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Ser Ser Phe Ser Gly     50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Thr Thr Pro Pro                 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg             100 105 <210> 11 <211> 118 <212> PRT <213> Artificial Sequence <220> <223> Synthetic polypeptide <400> 11 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Thr Ser Tyr             20 25 30 Tyr Ile Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val         35 40 45 Ala Glu Ile Ser Pro Tyr Ser Gly Ser Thr Tyr Tyr Ala Asp Ser Val     50 55 60 Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys                 85 90 95 Ala Leu Arg Ala Arg Pro Pro Ile Arg Leu Tyr Pro Arg Gly Ser Val             100 105 110 Met Asp Tyr Trp Gly Gln         115 <210> 12 <211> 108 <212> PRT <213> Artificial Sequence <220> <223> Synthetic polypeptide <400> 12 Asp Ile Gln Met Thr Gln Ser Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Ser Thr Ala             20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile         35 40 45 Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Ser Ser Phe Ser Gly     50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Thr Leu Pro Thr                 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg             100 105 <210> 13 <211> 118 <212> PRT <213> Artificial Sequence <220> <223> Synthetic polypeptide <400> 13 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Gly Tyr Tyr             20 25 30 Tyr Ile Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val         35 40 45 Ala Glu Ile Ser Pro Tyr Ser Gly Ser Thr Tyr Tyr Ala Asp Ser Val     50 55 60 Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys                 85 90 95 Ala Leu Arg Ala Arg Pro Pro Ile Arg Leu His Pro Arg Gly Ser Val             100 105 110 Met Asp Tyr Trp Gly Gln         115 <210> 14 <211> 108 <212> PRT <213> Artificial Sequence <220> <223> Synthetic polypeptide <400> 14 Asp Ile Gln Met Thr Gln Ser Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Ser Thr Ala             20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile         35 40 45 Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Ser Ser Phe Ser Gly     50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Thr Thr Pro Pro                 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg             100 105 <210> 15 <211> 118 <212> PRT <213> Artificial Sequence <220> <223> Synthetic polypeptide <400> 15 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Thr Asn Ser             20 25 30 Tyr Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val         35 40 45 Gly Trp Ile Thr Pro Tyr Gly Gly Tyr Thr Asn Tyr Ala Asp Ser Val     50 55 60 Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys                 85 90 95 Ala Arg Gly Ser Gly His Val Asn Ala Val Lys Asn Tyr Gly Tyr Val             100 105 110 Met Asp Tyr Trp Gly Gln         115 <210> 16 <211> 108 <212> PRT <213> Artificial Sequence <220> <223> Synthetic polypeptide <400> 16 Asp Ile Gln Met Thr Gln Ser Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Ser Thr Ala             20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile         35 40 45 Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Ser Ser Phe Ser Gly     50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Thr Thr Pro Pro                 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg             100 105 <210> 17 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Synthetic polypeptide <400> 17 Ser Tyr Tyr Ile Ser 1 5 <210> 18 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> Synthetic polypeptide <400> 18 Glu Ile Ser Pro Thyr Ser Gly Ser Thr Tyr Tyr Ala Asp Ser Val Lys 1 5 10 15 Gly      <210> 19 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> Synthetic polypeptide <400> 19 Arg Ala Arg Pro Pro Ile Arg Leu His Pro Arg Gly Ser Val 1 5 10 <210> 20 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Synthetic polypeptide <400> 20 Tyr Tyr Tyr Ile Ser 1 5 <210> 21 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> Synthetic polypeptide <400> 21 Arg Ala Arg Pro Pro Ile Arg Leu Tyr Pro Arg Gly Ser Val 1 5 10 <210> 22 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Synthetic polypeptide <400> 22 Asn Ser Tyr Ile His 1 5 <210> 23 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> Synthetic polypeptide <400> 23 Trp Ile Thr Pro Tyr Gly Gly Tyr Thr Asn Tyr Ala Asp Ser Val Lys 1 5 10 15 Gly      <210> 24 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> Synthetic polypeptide <400> 24 Gly Ser Gly His Val Asn Ala Val Lys Asn Tyr Gly Tyr Val 1 5 10 <210> 25 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Synthetic polypeptide <400> 25 Arg Ala Ser Gln Asp Val Ser Thr Ala Val Ala 1 5 10 <210> 26 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Synthetic polypeptide <400> 26 Ser Ala Ser Phe Leu Tyr Ser 1 5 <210> 27 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Synthetic polypeptide <400> 27 Gln Gln Ser Tyr Thr Thr Pro Pro Thr 1 5 <210> 28 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Synthetic polypeptide <400> 28 Gln Gln Ser Tyr Thr Leu Pro Thr Thr 1 5 <210> 29 <211> 1613 <212> PRT <213> Homo sapiens <400> 29 Met Gly Ala Val Leu Arg Ser Leu Leu Ala Cys Ser Phe Cys Val Leu 1 5 10 15 Leu Arg Ala Pro Leu Leu Leu Tyr Ala Asn Arg Arg Asp Leu Arg             20 25 30 Leu Val Asp Ala Thr Asn Gly Lys Glu Asn Ala Thr Ile Val Val Gly         35 40 45 Gly Leu Glu Asp Ala Ala Ala Val Asp Phe Val Phe Ser His Gly Leu     50 55 60 Ile Tyr Trp Ser Asp Val Ser Glu Glu Ala Ile Lys Arg Thr Glu Phe 65 70 75 80 Asn Lys Thr Glu Ser Val Gln Asn Val Val Val Ser Gly Leu Leu Ser                 85 90 95 Pro Asp Gly Leu Ala Cys Asp Trp Leu Gly Glu Lys Leu Tyr Trp Thr             100 105 110 Asp Ser Glu Thr Asn Arg Ile Glu Val Ser Asn Leu Asp Gly Ser Leu         115 120 125 Arg Lys Val Leu Phe Trp Gln Glu Leu Asp Gln Pro Arg Ala Ile Ala     130 135 140 Leu Asp Pro Ser Ser Gly Phe Met Tyr Trp Thr Asp Trp Gly Glu Val 145 150 155 160 Pro Lys Ile Glu Arg Ala Gly Met Asp Gly Ser Ser Arg Phe Ile Ile                 165 170 175 Ile Asn Ser Glu Ile Tyr Trp Pro Asn Gly Leu Thr Leu Asp Tyr Glu             180 185 190 Glu Gln Lys Leu Tyr Trp Ala Asp Ala Lys Leu Asn Phe Ile His Lys         195 200 205 Ser Asn Leu Asp Gly Thr Asn Arg Gln Ala Val Val Lys Gly Ser Leu     210 215 220 Pro His Phe Ala Leu Thr Leu Phe Glu Asp Ile Leu Tyr Trp Thr 225 230 235 240 Asp Trp Ser Thr His Ser Leu Ala Cys Asn Lys Tyr Thr Gly Glu                 245 250 255 Gly Leu Arg Glu Ile His Ser Asp Ile Phe Ser Pro Met Asp Ile His             260 265 270 Ala Phe Ser Gln Gln Arg Gln Pro Asn Ala Thr Asn Pro Cys Gly Ile         275 280 285 Asp Asn Gly Gly Cys Ser His Leu Cys Leu Met Ser Pro Val Lys Pro     290 295 300 Phe Tyr Gln Cys Ala Cys Pro Thr Gly Val Lys Leu Leu Glu Asn Gly 305 310 315 320 Lys Thr Cys Lys Asp Gly Ala Thr Glu Leu Leu Leu Ala Arg Arg                 325 330 335 Thr Asp Leu Arg Arg Ile Ser Leu Asp Thr Pro Asp Phe Thr Asp Ile             340 345 350 Val Leu Gln Leu Glu Asp Ile Arg His Ala Ile Ale Asp Tyr Asp         355 360 365 Pro Val Glu Gly Tyr Ile Tyr Trp Thr Asp Asp Glu Val Arg Ala Ile     370 375 380 Arg Arg Ser Phe Ile Asp Gly Ser Gly Ser Gln Phe Val Val Thr Ala 385 390 395 400 Gln Ile Ala His Pro Asp Gly Ile Ala Val Asp Trp Val Ala Arg Asn                 405 410 415 Leu Tyr Trp Thr Asp Thr Gly Thr Asp Arg Ile Glu Val Thr Arg Leu             420 425 430 Asn Gly Thr Met Arg Lys Ile Leu Ile Ser Glu Asp Leu Glu Glu Pro         435 440 445 Arg Ala Val Val Leu Asp Pro Met Val Gly Tyr Met Tyr Trp Thr Asp     450 455 460 Trp Gly Glu Ile Pro Lys Ile Glu Arg Ala Ala Leu Asp Gly Ser Asp 465 470 475 480 Arg Val Val Leu Val Asn Thr Ser Leu Gly Trp Pro Asn Gly Leu Ala                 485 490 495 Leu Asp Tyr Asp Glu Gly Lys Ile Tyr Trp Gly Asp Ala Lys Thr Asp             500 505 510 Lys Ile Glu Val Met Asn Thr Asp Gly Thr Gly Arg Arg Val Leu Val         515 520 525 Glu Asp Lys Ile Pro His Ile Phe Gly Phe Thr Leu Leu Gly Asp Tyr     530 535 540 Val Tyr Trp Thr Asp Trp Gln Arg Arg Ser Ser Glu Arg Val His Lys 545 550 555 560 Arg Ser Ala Glu Arg Glu Val Ile Ile Asp Gln Leu Pro Asp Leu Met                 565 570 575 Gly Leu Lys Ala Thr Asn Val His Arg Val Ile Gly Ser Asn Pro Cys             580 585 590 Ala Glu Glu Asn Gly Gly Cys Ser His Leu Cys Leu Tyr Arg Pro Gln         595 600 605 Gly Leu Arg Cys Ala Cys Pro Ile Gly Phe Glu Leu Ile Ser Asp Met     610 615 620 Lys Thr Cys Ile Val Pro Glu Ala Phe Leu Leu Phe Ser Arg Arg Ala 625 630 635 640 Asp Ile Arg Arg Ile Ser Leu Glu Thr Asn Asn Asn Asn Val Ala Ile                 645 650 655 Pro Leu Thr Gly Val Lys Glu Ala Ser Ala Leu Asp Phe Asp Val Thr             660 665 670 Asp Asn Arg Ile Tyr Trp Thr Asp Ile Ser Leu Lys Thr Ile Ser Arg         675 680 685 Ala Phe Met Asn Gly Ser Ala Leu Glu His Val Val Glu Phe Gly Leu     690 695 700 Asp Tyr Pro Glu Gly Met Ala Val Asp Trp Leu Gly Lys Asn Leu Tyr 705 710 715 720 Trp Ala Asp Thr Gly Thr Asn Arg Ile Glu Val Ser Lys Leu Asp Gly                 725 730 735 Gln His Arg Gln Val Leu Val Trp Lys Asp Leu Asp Ser Pro Arg Ala             740 745 750 Leu Ala Leu Asp Pro Ala Glu Gly Phe Met Tyr Trp Thr Glu Trp Gly         755 760 765 Gly Lys Pro Lys Ile Asp Arg Ala Ala Met Asp Gly Ser Glu Arg Thr     770 775 780 Thr Leu Val Pro Asn Val Gly Arg Ala Asn Gly Leu Thr Ile Asp Tyr 785 790 795 800 Ala Lys Arg Arg Leu Tyr Trp Thr Asp Leu Asp Thr Asn Leu Ile Glu                 805 810 815 Ser Ser Asn Met Leu Gly Leu Asn Arg Glu Val Ile Ala Asp Asp Leu             820 825 830 Pro His Phe Gly Leu Thr Gln Tyr Gln Asp Tyr Ile Tyr Trp Thr         835 840 845 Asp Trp Ser Arg Arg Ser Ile Glu Arg Ala Asn Lys Thr Ser Gly Gln     850 855 860 Asn Arg Thr Ile Gln Gly His Leu Asp Tyr Val Met Asp Ile Leu 865 870 875 880 Val Phe His Ser Ser Arg Gln Ser Gly Trp Asn Glu Cys Ala Ser Ser                 885 890 895 Asn Gly His Cys Ser His Leu Cys Leu Ala Val Pro Val Gly Gly Phe             900 905 910 Val Cys Gly Cys Pro Ala His Tyr Ser Leu Asn Ala Asp Asn Arg Thr         915 920 925 Cys Ser Ala Pro Thr Thr Phe Leu Leu Phe Ser Gln Lys Ser Ala Ile     930 935 940 Asn Arg Met Val Ile Asp Glu Gln Gln Ser Pro Asp Ile Ile Leu Pro 945 950 955 960 Ile His Ser Leu Arg Asn Val Arg Ala Ile Asp Tyr Asp Pro Leu Asp                 965 970 975 Lys Gln Leu Tyr Trp Ile Asp Ser Arg Gln Asn Met Ile Arg Lys Ala             980 985 990 Gln Glu Asp Gly Ser Gln Gly Phe Thr Val Val Val Ser Ser Val Pro         995 1000 1005 Ser Gln Asn Leu Glu Ile Gln Pro Tyr Asp Leu Ser Ile Asp Ile     1010 1015 1020 Tyr Ser Arg Tyr Ile Tyr Trp Thr Cys Glu Ala Thr Asn Val Ile     1025 1030 1035 Asn Val Thr Arg Leu Asp Gly Arg Ser Val Gly Val Val Leu Lys     1040 1045 1050 Gly Glu Gln Asp Arg Pro Arg Ala Val Val Asn Pro Glu Lys     1055 1060 1065 Gly Tyr Met Tyr Phe Thr Asn Leu Gln Glu Arg Ser Ser Lys Ile     1070 1075 1080 Glu Arg Ala Leu Asp Gly Thr Glu Arg Glu Val Leu Phe Phe     1085 1090 1095 Ser Gly Leu Ser Lys Pro Ile Ala Leu Ala Leu Asp Ser Arg Leu     1100 1105 1110 Gly Lys Leu Phe Trp Ala Asp Ser Asp Leu Arg Arg Ile Glu Ser     1115 1120 1125 Ser Asp Leu Ser Gly Ala Asn Arg Ile Val Leu Glu Asp Ser Asn     1130 1135 1140 Ile Leu Gln Pro Val Gly Leu Thr Val Phe Glu Asn Trp Leu Tyr     1145 1150 1155 Trp Ile Asp Lys Gln Gln Gln Met Ile Glu Lys Ile Asp Met Thr     1160 1165 1170 Gly Arg Glu Gly Arg Thr Lys Val Gln Ala Arg Ile Ala Gln Leu     1175 1180 1185 Ser Asp Ile His Ala Val Lys Glu Leu Asn Leu Gln Glu Tyr Arg     1190 1195 1200 Gln His Pro Cys Ala Gln Asp Asn Gly Gly Cys Ser His Ile Cys     1205 1210 1215 Leu Val Lys Gly Asp Gly Thr Thr Arg Cys Ser Cys Pro Met His     1220 1225 1230 Leu Val Leu Leu Gln Asp Glu Leu Ser Cys Gly Glu Pro Pro Thr     1235 1240 1245 Cys Ser Pro Gln Gln Phe Thr Cys Phe Thr Gly Glu Ile Asp Cys     1250 1255 1260 Ile Pro Val Ala Trp Arg Cys Asp Gly Phe Thr Glu Cys Glu Asp     1265 1270 1275 His Ser Asp Glu Leu Asn Cys Pro Val Cys Ser Glu Ser Gln Phe     1280 1285 1290 Gln Cys Ala Ser Gly Gln Cys Ile Asp Gly Ala Leu Arg Cys Asn     1295 1300 1305 Gly Asp Ala Asn Cys Gln Asp Lys Ser Asp Glu Lys Asn Cys Glu     1310 1315 1320 Val Leu Cys Leu Ile Asp Gln Phe Arg Cys Ala Asn Gly Gln Cys     1325 1330 1335 Ile Gly Lys His Lys Lys Cys Asp His Asn Val Asp Cys Ser Asp     1340 1345 1350 Lys Ser Asp Glu Leu Asp Cys Tyr Pro Thr Glu Glu Pro Ala Pro     1355 1360 1365 Gln Ala Thr Asn Thr Val Gly Ser Val Ile Gly Val Ile Val Thr     1370 1375 1380 Ile Phe Val Ser Gly Thr Val Tyr Phe Ile Cys Gln Arg Met Leu     1385 1390 1395 Cys Pro Arg Met Lys Gly Asp Gly Glu Thr Met Thr Asn Asp Tyr     1400 1405 1410 Val Val His Gly Pro Ala Ser Val Pro Leu Gly Tyr Val Pro His     1415 1420 1425 Pro Ser Ser Leu Ser Gly Ser Leu Pro Gly Met Ser Arg Gly Lys     1430 1435 1440 Ser Met Ile Ser Ser Leu Ser Ile Met Gly Gly Ser Ser Gly Pro     1445 1450 1455 Pro Tyr Asp Arg Ala His Val Thr Gly Ala Ser Ser Ser Ser Ser     1460 1465 1470 Ser Ser Thr Lys Gly Thr Tyr Phe Pro Ala Ile Leu Asn Pro Pro     1475 1480 1485 Pro Ser Ala Thr Glu Arg Ser His Tyr Thr Met Glu Phe Gly     1490 1495 1500 Tyr Ser Ser Asn Ser Ser Ser Thr His Arg Ser Tyr Ser Tyr Arg     1505 1510 1515 Pro Tyr Ser Tyr Arg His Phe Ala Pro Pro Thr Thr Pro Cys Ser     1520 1525 1530 Thr Asp Val Cys Asp Ser Asp Tyr Ala Pro Ser Arg Arg Met Thr     1535 1540 1545 Ser Val Ala Thr Ala Lys Gly Tyr Thr Ser Asp Leu Asn Tyr Asp     1550 1555 1560 Ser Glu Pro Val Pro Pro Pro Pro Thr Pro Arg Ser Gln Tyr Leu     1565 1570 1575 Ser Ala Glu Glu Asn Tyr Glu Ser Cys Pro Ser Ser Pro Tyr Thr     1580 1585 1590 Glu Arg Ser Tyr Ser His His Leu Tyr Pro Pro Pro Ser Ser Pro     1595 1600 1605 Cys Thr Asp Ser Ser     1610 <210> 30 <211> 890 <212> PRT <213> Artificial Sequence <220> <223> Synthetic polypeptide <400> 30 Met Gly Gly Thr Ala Ala Arg Leu Gly Ala Val Ile Leu Phe Val Val 1 5 10 15 Ile Val Gly Leu His Gly Val Arg Gly Lys Gly Ser Ala Ala Pro Leu             20 25 30 Leu Leu Tyr Ala Asn Arg Asp Leu Arg Leu Val Asp Ala Thr Asn         35 40 45 Gly Lys Glu Asn Ala Thr Ile Val Val Gly Gly Leu Glu Asp Ala Ala     50 55 60 Ala Val Asp Phe Val Phe Ser His Gly Leu Ile Tyr Trp Ser Asp Val 65 70 75 80 Ser Glu Glu Ala Ile Lys Arg Thr Glu Phe Asn Lys Thr Glu Ser Val                 85 90 95 Gln Asn Val Val Val Ser Gly Leu Leu Ser Pro Asp Gly Leu Ala Cys             100 105 110 Asp Trp Leu Gly Glu Lys Leu Tyr Trp Thr Asp Ser Glu Thr Asn Arg         115 120 125 Ile Glu Val Ser Asn Leu Asp Gly Ser Leu Arg Lys Val Leu Phe Trp     130 135 140 Gln Glu Leu Asp Gln Pro Arg Ala Ile Ala Leu Asp Pro Ser Ser Gly 145 150 155 160 Phe Met Tyr Trp Thr Asp Trp Gly Glu Val Pro Lys Ile Glu Arg Ala                 165 170 175 Gly Met Asp Gly Ser Ser Arg Phe Ile Ile Ile Asn Ser Glu Ile Tyr             180 185 190 Trp Pro Asn Gly Leu Thr Leu Asp Tyr Glu Glu Gln Lys Leu Tyr Trp         195 200 205 Ala Asp Ala Lys Leu Asn Phe Ile His Lys Ser Asn Leu Asp Gly Thr     210 215 220 Asn Arg Gln Ala Val Val Lys Gly Ser Leu Pro His Pro Phe Ala Leu 225 230 235 240 Thr Leu Phe Glu Asp Ile Leu Tyr Trp Thr Asp Trp Ser Thr His Ser                 245 250 255 Ile Leu Ala Cys Asn Lys Tyr Thr Gly Glu Gly Leu Arg Glu Ile His             260 265 270 Ser Asp Ile Phe Ser Pro Met Asp Ile His Ala Phe Ser Gln Gln Arg         275 280 285 Gln Pro Asn Ala Thr Asn Pro Cys Gly Ile Asp Asn Gly Gly Cys Ser     290 295 300 His Leu Cys Leu Met Ser Pro Val Lys Pro Phe Tyr Gln Cys Ala Cys 305 310 315 320 Pro Thr Gly Val Lys Leu Leu Glu Asn Gly Lys Thr Cys Lys Asp Gly                 325 330 335 Ala Thr Glu Leu Leu Leu Ala Arg Arg Thr Asp Leu Arg Arg Ile             340 345 350 Ser Leu Asp Thr Pro Asp Phe Thr Asp Ile Val Leu Gln Leu Glu Asp         355 360 365 Ile Arg His Ala Ile Ale Ile Asp Tyr Asp Pro Val Glu Gly Tyr Ile     370 375 380 Tyr Trp Thr Asp Asp Glu Val Arg Ala Ile Arg Arg Ser Phe Ile Asp 385 390 395 400 Gly Ser Gly Ser Gln Phe Val Val Thr Ala Gln Ile Ala His Pro Asp                 405 410 415 Gly Ile Ala Val Asp Trp Val Ala Arg Asn Leu Tyr Trp Thr Asp Thr             420 425 430 Gly Thr Asp Arg Ile Glu Val Thr Arg Leu Asn Gly Thr Met Arg Lys         435 440 445 Ile Leu Ile Ser Glu Asp Leu Glu Glu Pro Arg Ile Val Leu Asp     450 455 460 Pro Met Val Gly Tyr Met Tyr Trp Thr Asp Trp Gly Glu Ile Pro Lys 465 470 475 480 Ile Glu Arg Ala Leu Asp Gly Ser Asp Arg Val Val Leu Val Asn                 485 490 495 Thr Ser Leu Gly Trp Pro Asn Gly Leu Ala Leu Asp Tyr Asp Glu Gly             500 505 510 Lys Ile Tyr Trp Gly Asp Ala Lys Thr Asp Lys Ile Glu Val Met Asn         515 520 525 Thr Asp Gly Thr Gly Arg Arg Val Leu Val Glu Asp Lys Ile Pro His     530 535 540 Ile Phe Gly Phe Thr Leu Leu Gly Asp Tyr Val Tyr Trp Thr Asp Trp 545 550 555 560 Gln Arg Arg Ser Ser Glu Arg Val His Lys Arg Ser Ala Glu Arg Glu                 565 570 575 Val Ile Ile Asp Gln Leu Pro Asp Leu Met Gly Leu Lys Ala Thr Asn             580 585 590 Val His Arg Val Ile Gly Ser Asn Pro Cys Ala Glu Glu Asn Gly Gly         595 600 605 Cys Ser His Leu Cys Leu Tyr Arg Pro Gln Gly Leu Arg Cys Ala Cys     610 615 620 Pro Ile Gly Phe Glu Leu Ile Ser Asp Met Lys Thr Cys Ile Val Pro 625 630 635 640 Glu Ala Phe Leu Leu Phe Ser Arg Arg Ala Asp Ile Arg Arg Leu Glu                 645 650 655 Ser Gly Gly Gly Gly Val Thr Asp Lys Thr His Thr Cys Pro Pro Cys             660 665 670 Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro         675 680 685 Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys     690 695 700 Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp 705 710 715 720 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu                 725 730 735 Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu             740 745 750 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn         755 760 765 Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly     770 775 780 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu 785 790 795 800 Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr                 805 810 815 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn             820 825 830 Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe         835 840 845 Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn     850 855 860 Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 865 870 875 880 Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys                 885 890 <210> 31 <211> 882 <212> PRT <213> Artificial Sequence <220> <223> Synthetic polypeptide <400> 31 Met Gly Gly Thr Ala Ala Arg Leu Gly Ala Val Ile Leu Phe Val Val 1 5 10 15 Ile Val Gly Leu His Gly Val Arg Gly Lys Gly Ser Val Pro Glu Ala             20 25 30 Phe Leu Leu Phe Ser Arg Arg Ala Asp Ile Arg Arg Ile Ser Leu Glu         35 40 45 Thr Asn Asn Asn Val Ala Ile Pro Leu Thr Gly Val Lys Glu Ala     50 55 60 Ser Ala Leu Asp Phe Asp Val Thr Asp Asn Arg Ile Tyr Trp Thr Asp 65 70 75 80 Ile Ser Leu Lys Thr Ile Ser Arg Ala Phe Met Asn Gly Ser Ala Leu                 85 90 95 Glu His Val Glu Phe Gly Leu Asp Tyr Pro Glu Gly Met Ala Val             100 105 110 Asp Trp Leu Gly Lys Asn Leu Tyr Trp Ala Asp Thr Gly Thr Asn Arg         115 120 125 Ile Glu Val Ser Lys Leu Asp Gly Gln His Arg Gln Val Leu Val Trp     130 135 140 Lys Asp Leu Asp Ser Pro Arg Ala Leu Ala Leu Asp Pro Ala Glu Gly 145 150 155 160 Phe Met Tyr Trp Thr Glu Trp Gly Gly Lys Pro Lys Ile Asp Arg Ala                 165 170 175 Ala Met Asp Gly Ser Glu Arg Thr Thr Leu Val Pro Asn Val Gly Arg             180 185 190 Ala Asn Gly Leu Thr Ile Asp Tyr Ala Lys Arg Arg Leu Tyr Trp Thr         195 200 205 Asp Leu Asp Thr Asn Leu Ile Glu Ser Ser Asn Met Leu Gly Leu Asn     210 215 220 Arg Glu Val Ile Ala Asp Asp Leu Pro His Pro Phe Gly Leu Thr Gln 225 230 235 240 Tyr Gln Asp Tyr Ile Tyr Trp Thr Asp Trp Ser Arg Arg Ser Ile Glu                 245 250 255 Arg Ala Asn Lys Thr Ser Gly Gln Asn Arg Thr Ile Ile Gln Gly His             260 265 270 Leu Asp Tyr Val Met Asp Ile Leu Val Phe His Ser Ser Arg Gln Ser         275 280 285 Gly Trp Asn Glu Cys Ala Ser Ser Asn Gly His Cys Ser His Leu Cys     290 295 300 Leu Ala Val Pro Val Gly Gly Phe Val Cys Gly Cys Pro Ala His Tyr 305 310 315 320 Ser Leu Asn Ala Asp Asn Arg Thr Cys Ser Ala Pro Thr Thr Phe Leu                 325 330 335 Leu Phe Ser Gln Lys Ser Ala Ile Asn Arg Met Val Ile Asp Glu Gln             340 345 350 Gln Ser Pro Asp Ile Ile Leu Pro Ile His Ser Leu Arg Asn Val Arg         355 360 365 Ala Ile Asp Tyr Asp Pro Leu Asp Lys Gln Leu Tyr Trp Ile Asp Ser     370 375 380 Arg Gln Asn Met Ile Arg Lys Ala Gln Glu Asp Gly Ser Gln Gly Phe 385 390 395 400 Thr Val Val Val Ser Ser Val Ser Ser Gln Asn Leu Glu Ile Gln Pro                 405 410 415 Tyr Asp Leu Ser Ile Asp Ile Tyr Ser Arg Tyr Ile Tyr Trp Thr Cys             420 425 430 Glu Ala Thr Asn Val Ile Asn Val Thr Arg Leu Asp Gly Arg Ser Val         435 440 445 Gly Val Val Leu Lys Gly Glu Gln Asp Arg Pro Arg Ala Val Val     450 455 460 Asn Pro Glu Lys Gly Tyr Met Tyr Phe Thr Asn Leu Gln Glu Arg Ser 465 470 475 480 Pro Lys Ile Glu Arg Ala Leu Asp Gly Thr Glu Arg Glu Val Leu                 485 490 495 Phe Phe Ser Gly Leu Ser Lys Pro Ile Ala Leu Ala Leu Asp Ser Arg             500 505 510 Leu Gly Lys Leu Phe Trp Ala Asp Ser Asp Leu Arg Arg Ile Glu Ser         515 520 525 Ser Asp Leu Ser Gly Ala Asn Arg Ile Val Leu Glu Asp Ser Asn Ile     530 535 540 Leu Gln Pro Val Gly Leu Thr Val Phe Glu Asn Trp Leu Tyr Trp Ile 545 550 555 560 Asp Lys Gln Gln Gln Met Ile Glu Lys Ile Asp Met Thr Gly Arg Glu                 565 570 575 Gly Arg Thr Lys Val Gln Ala Arg Ile Ala Gln Leu Ser Asp Ile His             580 585 590 Ala Val Lys Glu Leu Asn Leu Gln Glu Tyr Arg Gln His Pro Cys Ala         595 600 605 Gln Asp Asn Gly Gly Cys Ser His Ile Cys Leu Val Lys Gly Asp Gly     610 615 620 Thr Thr Arg Cys Ser Cys Pro Met His Leu Val Leu Leu Gln Asp Glu 625 630 635 640 Leu Ser Cys Gly Thr Gly Leu Glu Ser Gly Gly Gly Gly Val Thr Asp                 645 650 655 Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly             660 665 670 Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile         675 680 685 Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu     690 695 700 Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His 705 710 715 720 Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg                 725 730 735 Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys             740 745 750 Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu         755 760 765 Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr     770 775 780 Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu 785 790 795 800 Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp                 805 810 815 Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val             820 825 830 Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp         835 840 845 Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His     850 855 860 Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 865 870 875 880 Gly Lys          <210> 32 <211> 23 <212> DNA <213> Artificial Sequence <220> Synthetic sequence <400> 32 aatgctgctg aactgaatag aaa 23 <210> 33 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 33 aaccggtcct agcgaaaa 18 <210> 34 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 34 ccgagcactg tttcaaatct ccca 24 <210> 35 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 35 tgagagtgtg acattgttgg aa 22 <210> 36 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 36 gtaaaatctg tgtgcaatta tcatgt 26 <210> 37 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 37 aatcattgaa aatgactaac acaagaccct gtaaat 36 <210> 38 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 38 tgaggacgca ggagtgaa 18 <210> 39 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 39 cccagagagt ggccaaat 18 <210> 40 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 40 cctgtttgct gccacccatg a 21 <210> 41 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> Synthetic polypeptide <220> <221> MISC_FEATURE <222> (2) (2) <223> X is any amino acid <220> <221> MISC_FEATURE &Lt; 222 > (3) <223> X is I or V <400> 41 Asn Xaa Xaa Lys One <210> 42 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Synthetic polypeptide <220> <221> MISC_FEATURE <222> (2) (2) <223> X is any amino acid <220> <221> MISC_FEATURE &Lt; 222 > (3) <223> X is I or V <400> 42 Asn Xaa Xaa Lys Asn 1 5 <210> 43 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> Synthetic polypeptide <220> <221> MISC_FEATURE <222> (2) (2) <223> X is any amino acid <400> 43 Asn Xaa Val Lys One <210> 44 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> Synthetic polypeptide <220> <221> MISC_FEATURE <222> (2) (2) <223> X is any amino acid <400> 44 Asn Xaa Ile Lys One <210> 45 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Synthetic polypeptide <220> <221> MISC_FEATURE <222> (2) (2) <223> X is any amino acid <400> 45 Asn Xaa Val Lys Asn 1 5 <210> 46 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Synthetic polypeptide <220> <221> MISC_FEATURE <222> (2) (2) <223> X is any amino acid <400> 46 Asn Xaa Ile Lys Asn 1 5 <210> 47 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> Synthetic polypeptide <400> 47 Asn Ala Val Lys One <210> 48 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> Synthetic polypeptide <400> 48 Asn Ala Ile Lys One <210> 49 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Synthetic polypeptide <400> 49 Asn Ala Val Lys Asn 1 5 <210> 50 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Synthetic polypeptide <400> 50 Asn Ala Ile Lys Asn 1 5 <210> 51 <211> 116 <212> PRT <213> Artificial Sequence <220> <223> Synthetic polypeptide <400> 51 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Thr Gly Tyr             20 25 30 Trp Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val         35 40 45 Gly Thr Ile Ser Pro Ala Gly Gly Ser Thr Asp Tyr Ala Asp Ser Val     50 55 60 Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys                 85 90 95 Ala Arg Thr Asp Trp Arg Phe His His Ala Gly Glu Tyr Ala Met Asp             100 105 110 Tyr Trp Gly Gln         115 <210> 52 <211> 115 <212> PRT <213> Artificial Sequence <220> <223> Synthetic polypeptide <400> 52 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Thr Asp Tyr             20 25 30 Trp Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val         35 40 45 Ala Gly Ile Ser Pro Asp Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val     50 55 60 Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys                 85 90 95 Ala Arg Trp Ser Tyr Ile Ser Arg Tyr Phe Ser Ser Val Met Asp Tyr             100 105 110 Trp Gly Gln         115 <210> 53 <211> 113 <212> PRT <213> Artificial Sequence <220> <223> Synthetic polypeptide <400> 53 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Thr Asn Tyr             20 25 30 Asp Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val         35 40 45 Ala Ser Ile Tyr Pro Ala Gly Gly Asp Thr Asp Tyr Ala Asp Ser Val     50 55 60 Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys                 85 90 95 Ala Arg Ser Pro Gly Trp Ala Leu Arg Gly Ala Met Asp Tyr Trp Gly             100 105 110 Gln      <210> 54 <211> 110 <212> PRT <213> Artificial Sequence <220> <223> Synthetic polypeptide <400> 54 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ser Ser Gly Phe Thr Phe Thr Gly Asn             20 25 30 Asp Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val         35 40 45 Gly Arg Ile Tyr Pro Tyr Gly Gly Tyr Thr Asp Tyr Ala Asp Ser Val     50 55 60 Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys                 85 90 95 Ala Lys Glu Val Thr Tyr His Leu Phe Asp Tyr Trp Gly Gln             100 105 110 <210> 55 <211> 115 <212> PRT <213> Artificial Sequence <220> <223> Synthetic polypeptide <400> 55 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Gly Ser             20 25 30 Ser Ile Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val         35 40 45 Gly Val Ile Ser Ser Ser Gly Ala Thr Tyr Tyr Ala Asp Ser Val     50 55 60 Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys                 85 90 95 Ala Arg Trp Pro Ala Gly Ala Phe Leu Gly Tyr Tyr Gly Met Asp Tyr             100 105 110 Trp Gly Gln         115 <210> 56 <211> 118 <212> PRT <213> Artificial Sequence <220> <223> Synthetic polypeptide <400> 56 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Gly Phe Tyr             20 25 30 Tyr Leu Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val         35 40 45 Ala Glu Ile Ser Pro Tyr Ser Gly Ser Thr Tyr Tyr Ala Asp Ser Val     50 55 60 Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys                 85 90 95 Ala Leu Arg Ala Arg Pro Pro Ile Arg Leu His Pro Arg Gly Ser Val             100 105 110 Met Asp Tyr Trp Gly Gln         115 <210> 57 <211> 118 <212> PRT <213> Artificial Sequence <220> <223> Synthetic polypeptide <400> 57 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Gly Phe Tyr             20 25 30 Tyr Ile Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val         35 40 45 Ala Glu Ile Ser Pro Tyr Ser Gly Ser Thr Tyr Tyr Ala Asp Ser Val     50 55 60 Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys                 85 90 95 Ala Leu Arg Ala Arg Pro Pro Ile Arg Leu His Pro Arg Gly Ser Val             100 105 110 Met Asp Tyr Trp Gly Gln         115 <210> 58 <211> 108 <212> PRT <213> Artificial Sequence <220> <223> Synthetic polypeptide <400> 58 Asp Ile Gln Met Thr Gln Ser Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Ser Thr Ala             20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile         35 40 45 Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Ser Ser Phe Ser Gly     50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Ala Ile Pro Thr                 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg             100 105

Claims (76)

  1. (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 20;
    (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 18; And
    (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 19
    A second VH domain comprising a second VH domain,
    (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 25;
    (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 26; And
    (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 27
    A first VL domain comprising a first VL domain,
    (g) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 22;
    (h) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 23; And
    (i) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 24
    A second VH domain comprising &lt; RTI ID = 0.0 &gt;
    (j) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 25;
    (k) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 26; And
    (1) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 27
    Lt; RTI ID = 0.0 &gt;
    , Or
    (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 17;
    (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 18; And
    (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 19
    A second VH domain comprising a second VH domain,
    (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 25;
    (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 26; And
    (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 27
    A first VL domain comprising a first VL domain,
    (g) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 22;
    (h) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 23; And
    (i) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 24
    A second VH domain comprising &lt; RTI ID = 0.0 &gt;
    (j) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 25;
    (k) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 26; And
    (1) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 27
    Lt; RTI ID = 0.0 &gt;
    , Or
    (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 20;
    (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 18; And
    (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 21
    A second VH domain comprising a second VH domain,
    (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 25;
    (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 26; And
    (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 27
    A first VL domain comprising a first VL domain,
    (g) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 22;
    (h) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 23; And
    (i) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 24
    A second VH domain comprising &lt; RTI ID = 0.0 &gt;
    (j) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 25;
    (k) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 26; And
    (1) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 27
    Lt; RTI ID = 0.0 &gt;
    / RTI &gt;
    An isolated bispecific antibody that binds to two different regions of LRP6.
  2. The method according to claim 1,
    (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 20;
    (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 18; And
    (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 19
    A second VH domain comprising a second VH domain,
    (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 25;
    (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 26; And
    (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 27
    A first VL domain comprising a first VL domain,
    (g) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 22;
    (h) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 23; And
    (i) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 24
    A second VH domain comprising &lt; RTI ID = 0.0 &gt;
    (j) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 25;
    (k) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 26; And
    (1) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 27
    Lt; RTI ID = 0.0 &gt;
    Lt; / RTI &gt; antibody.
  3. The method according to claim 1,
    (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 17;
    (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 18; And
    (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 19
    A second VH domain comprising a second VH domain,
    (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 25;
    (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 26; And
    (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 27
    A first VL domain comprising a first VL domain,
    (g) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 22;
    (h) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 23; And
    (i) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 24
    A second VH domain comprising &lt; RTI ID = 0.0 &gt;
    (j) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 25;
    (k) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 26; And
    (1) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 27
    Lt; RTI ID = 0.0 &gt;
    Lt; / RTI &gt; antibody.
  4. The method according to claim 1,
    (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 20;
    (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 18; And
    (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 21
    A second VH domain comprising a second VH domain,
    (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 25;
    (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 26; And
    (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 27
    A first VL domain comprising a first VL domain,
    (g) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 22;
    (h) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 23; And
    (i) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 24
    A second VH domain comprising &lt; RTI ID = 0.0 &gt;
    (j) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 25;
    (k) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 26; And
    (1) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 27
    Lt; RTI ID = 0.0 &gt;
    Lt; / RTI &gt; antibody.
  5. The method according to claim 1,
    (a) a first VH comprising the amino acid sequence of SEQ ID NO: 13;
    (b) a first VL comprising the amino acid sequence of SEQ ID NO: 14;
    (c) a second VH comprising the amino acid sequence of SEQ ID NO: 15; And
    (d) a second VL comprising the amino acid sequence of SEQ ID NO: 16
    , Or
    (a) a first VH comprising the amino acid sequence of SEQ ID NO: 9;
    (b) a first VL comprising the amino acid sequence of SEQ ID NO: 10;
    (c) a second VH comprising the amino acid sequence of SEQ ID NO: 15; And
    (d) a second VL comprising the amino acid sequence of SEQ ID NO: 16
    , Or
    (a) a first VH comprising the amino acid sequence of SEQ ID NO: 11;
    (b) a first VL comprising the amino acid sequence of SEQ ID NO: 12;
    (c) a second VH comprising the amino acid sequence of SEQ ID NO: 15; And
    (d) a second VL comprising the amino acid sequence of SEQ ID NO: 16
    Lt; / RTI &gt; antibody.
  6. An antibody that competes for binding to the LRP6 with the bispecific antibody of any one of claims 1 to 5.
  7. An antibody that binds to the same two epitopes as the bispecific antibody of any one of claims 1 to 5.
  8. 8. The antibody of claim 7, wherein one of the two epitopes comprises the amino acid residues R28, E51, D52, V70, S71, E73, L95, S96, D98, E115, R141 and N185 of LRP6 (SEQ ID NO: 29).
  9. 9. The antibody of claim 8, wherein one of the two epitopes further comprises R29, W188, K202, P225, H226, S243 and F266 of LRP6 (SEQ ID NO: 29).
  10. The bispecific antibody according to any one of claims 1 to 5, which is a monoclonal antibody.
  11. 6. A bispecific antibody according to any one of claims 1 to 5, which is a chimeric, human or humanized antibody.
  12. An isolated nucleic acid encoding the bispecific antibody of any one of claims 1 to 5.
  13. 12. A host cell comprising the nucleic acid of claim 12.
  14. An immunoconjugate comprising a bispecific antibody of any one of claims 1 to 5 and a cytotoxic agent.
  15. An immunoconjugate comprising a bispecific antibody of any one of claims 1 to 5 and a pharmaceutically acceptable carrier or a bispecific antibody of any one of claims 1 to 5 and a cytotoxic agent &Lt; / RTI &gt; and a pharmaceutically acceptable carrier.
  16. delete
  17. A pharmaceutical composition for use in the treatment of cancer, comprising the bispecific antibody of any one of claims 1 to 5.
  18. 18. The pharmaceutical composition according to claim 17, wherein the cancer is selected from the group consisting of non-small cell lung cancer, breast cancer, pancreatic cancer, ovarian cancer, kidney cancer and prostate cancer.
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